Proanthocyanidin polymers having antiviral activity and methods of obtaining same

ABSTRACT

The present invention provides for proanthocyanidin polymers with significant antiviral activity. The proanthocyanidin polymers can be chemically synthesized or can be isolated from a Croton or a Calophyllum plant species. The present invention encompasses methods of using proanthocyanidin polymers in treating warm-blooded animals, including humans, infected with paramyxovaridae such as respiratory syncytial virus, orthomyxovaridae such as influenza A, B and C, and herpes viruses such as Herpes Simplex virus.

CROSS-REFERENCE TO RELATED APPLICATION

This is a continuation-in-part of abandoned application Ser. No.07/596,893, filed Oct. 12, 1990, which is incorporated herein byreference.

1. FIELD OF THE INVENTION

The present invention relates to the use of proanthocyanidin polymers,having 2 to 30 flavonoid units, in treating respiratory virusinfections. Additionally, it has been found that the use of theproanthocyanidin polymers having 6 to 30 flavonoid units is effective intreating virus infections in general. The chemical characteristics ofcertain newly discovered proanthocyanidin polymers are also encompassed.

2. BACKGROUND OF THE INVENTION 2.1 ETHNOBOTANICAL USES OF EXTRACTS FROMTHE CROTON TREE AND FROM CALOPHYLLUM INOPHYLUM

A number of different Croton tree species, including Croton sakutaris,Croton gossypifolius, Croton palanostima, Croton lechleri, Crotonerythrochilus and Croton draconoides, found in South America, produce ared viscous latex sap called Sangre de Drago. It is most often utilizedby mixed descent and native people of the Peruvian Amazon for flu anddiarrhea. It is taken internally for tonsillitis, throat infections,tuberculosis, peptic ulcers, intestinal disorders, rheumatism and toenhance fertility and is used by both adults and children. It is alsoused extensively to stop bleeding, herpes, and for wound healing. Thesap is placed directly on open wounds as an anti-infective and toaccelerate the healing process. It is applied to the gums of patientsafter tooth extractions. It is also utilized as a vaginal wash in casesof excessive bleeding.

It has been shown that Sangre de Drago from Croton draconoides and fromCroton lechleri contains an alkaloid identified as taspine, whichexhibits anti-inflammatory activity, Persinos et al., 1979, J. Pharm.Sci., 68:124. Taspine has also been shown to inhibit RNA-directed DNApolymerase activity in the myeloblastosis virus, Rauscher leukemia virusand Simian sarcoma virus (Sethi, 1977, Canadian J. Pharm. Sci., 12:7).

Calophyllum inophylum is a tree ranging from India to East Africa toPolynesia. Seed oil is used in folk medicine as an antiparasitic intreatment of scabies, ringworm and dermatosis as well as other uses suchas for analgesia. In Indo-China the powdered resin is used for ulcersand wound healing. In Indonesia the bark is applied externally to treatswollen glands and internally as a diuretic. The sap is used as anemollient for chest pain as well as for tumors and swelling. Leafextracts are used as a wash for inflamed eyes. The Cambodians use leafextracts in inhalations for treatment of vertigo and migraine. TheSamoans use the sap as an arrow poison.

2.2 PROANTHOCYANIDIN MONOMERS AND POLYMERS AND THEIR USES

Proanthocyanidin and proanthocyanidin polymers are found as colorlessphenolic substances in a wide variety of many plants, particularly thosewith a woody habit of growth (e.g., the Croton species and Calophylluminophylum). The general chemical structure of a polymericproanthocyanidin consists of linear chains of 5, 7, 3', 4' tetrahydroxyor 5, 7, 3', 5' pentahydroxy flavonoid 3-ol units linked togetherthrough common C(4)-(6) and/or C(4)-C(8) bonds, as shown below. ##STR1##Biosynthetic studies have indicated that proanthocyanidin polymersconsist of monomer units of the type shown below, See Fletcher et al.,1977, J.C.S. Perkin, 1:1628. ##STR2##

The monomer unit (generally termed "leucoanthocyanidin") of the polymerchain may be based on either of two stereochemistries of the C-ring, atthe 2 and/or 4 position designated cis (called epicatechins) or trans(called catechin) Therefore, the polymer chains are based on differentstructural units, which create a wide variation of polymericproanthocyanidins and a large number of possible isomers (Hemingway etal., 1982, J.C.S. in, 1:1217). C13 NMR has been useful to identify thestructures of polymeric proanthocyanidins and recent work has elucidatedthe chemistry of di-, tri- and tetra-meric proanthocyanidins. Largerpolymers of the flavonoid 3-ol units are predominant in most plants, andare found with average molecular weights above 2,000 daltons, containing6 or more units, Newman et al., 1987, Mag. Res. Chem., 25:118.

Proanthocyanidins have been reported to possess protein binding abilityand a possible biological role (Newman et al., 1987, Mag. Res. Chem.,25:118). Proanthocyanidin monomers and dimers have been used in thetreatment of diseases associated with increased capillary fragility andhave also been shown to have anti-inflammatory effects in experimentalanimals (Beladi et al., 1977, Ann. N.Y. Acad. Sci., 284:358). Aprocyanidin monomer was found to have antiviral activity against HerpesSimplex virus in an in vitro assay. (Beladi, et al., supra.) Beladi etal. tested the viricidal effect of a number of flavonoid monomersincluding apigenin, pelargonidin, quercetin and a proanthocyanidinmonomer on Herpes Simplex virus. The proanthocyanidin monomer was foundto be the most effective in virus-inactivating activity, followed bypelargonidin and quercetin. Apigenin had only a slight effect. Theeffect of quercetin and the procyanidin monomer on the multiplication ofHerpes Simplex virus in HEp-2 cells was also investigated. As concludedby Beladi et al., the monomeric flavonoids tested had viricidalactivity, but only a slight inhibitory effect on virus multiplication inthe in vitro assays.

Prior to the present invention, there has been no disclosure regardingthe use of proanthocyanidins containing two or more flavonoid units fortreating respiratory virus infections. Additionally, there has been nodisclosure regarding the use of proanthocyanidins of six or moreflavonoid units for treating virus infections in general.

For the purpose of the present application, a proanthocyanidin polymerisolated from the Croton species is designated "proanthocyanidin polymerA" and that isolated from Calophyllum inophylum is designated"proanthocyanidin polymer B". However, such designation is solely forsimplicity of discussion and not necessarily intended to imply that thepolymers are different classes of compounds. Both proanthocyanidinpolymer A and proanthocyanidin polymer B are considered to be within theterm proanthocyanidin polymer as described herein.

2.3 RESPIRATORY SYNCYTIAL VIRUS

Respiratory Syncytial Virus (RSV) belongs to the virus familyParamyxoviridae, genus Pneumovirus, and is responsible for causing lowerrespiratory tract infections such as bronchiolitis and pneumonia in theinfant and child. The virus is considered to be in important agent ofacute respiratory disease in children and it is expected that up to 50%of infants will suffer from RSV infections during their first winter. Inyoung children with pulmonary or cardiac disease, up to a 37% mortalityrate has been reported due to RSV. Although RSV respiratory infection iscommon in the young, the virus is found in respiratory secretions ofinfected persons of any age. Outbreaks among the elderly have beenassociated with serious and fatal illness, and RSV infection can be thesource of fever and pulmonary infiltrates in immunosuppressed adults(Englund et al., 1988, Ann. Int. Med.,1:203).

A vaccine of inactivated RSV has proven to be ineffective against RSVinfection and leads to an unusual immune and lung inflammation uponsubsequent RSV infection. Therefore, recent research has concentrated ondeveloping either potent live vaccines or an effective antiviralcompound.

Ribavirin, (1-β-D-ribofuranosyl-1, 2, 4-triazole 3-carboxamide), thecurrent drug of choice, has been found to reduce the severity of illnessand amount of virus shed in acute respiratory infections of RSV orinfluenza virus. Currently ribavirin is used for the treatment of RSVinfections of the respiratory tract in aerosol form. However, because ofits toxicity, ribavirin is less desirable for use systemically.Additionally, ribavirin has been found to be teratogenic in animals.This has raised serious concern for female health care personneladministering or exposed to ribavirin (Gladu et al., 1989, J. Toxic Env.Health, 28:1).

2.4 OTITIS MEDIA AND OTITIS EXTERNA

Otitis media, inflammation of the middle ear, is second only to colds asthe most common disease of early childhood. More than 60% of allchildren will have an episode of otitis media by age six. The greatestrisk for the development of acute otitis media lies in infections causedby respiratory syncytial viruses, rhinoviruses, influenza A viruses oradenoviruses (Henderson, et al., N. Eng. J. Med., 1982, 1377; Ruuskanin,et al., Pediatr. Infect. Dis. J., 1989, 94; Sanyai, et al., J. Pediatr.,1980, 11). It has been found that prevention of the respiratory viralinfection decreases the incidence of otitis media (Heikkinen, et al.,AJDC, 445 (1991). Use of a flu vaccine has been found to be effective.However, there are a number of reasons why this is an unsatisfactoryapproach: it is necessary to administer the vaccine annually, two dosesmust be given initially, there is a problem of continual antigenicvariation of the influenza viruses and the resulting fluctuation inefficacy of the vaccine and the cost.

2.5 INFLUENZA VIRUSES

Influenza viruses are members of the Orthomyxoviridae family and aredistributed world-wide. Five pandemics have occurred in the 20thcentury, with the 1918 outbreak killing at least 21 million. In theUnited States, over 500,000 deaths have been attributed to influenzaepidemics over the last twenty years. The outbreaks are seasonal,occurring almost every winter, and transmission is primarily through therespiratory route. Amantadine has been shown to shorten the feverduration and respiratory symptoms by only about 50%, but causes somecentral nervous system side effects. Rimantidine, as yet unlicensed,shows a similar effect, although it is not associated with the centralnervous system side-effects seen with amantadine. Primary influenzaviral pneumonia, a common complication of influenza infections, has notbeen shown to be treatable by either rimantidine or amantadine.Prophylactically, influenza has been controlled by the use ofinactivated influenza virus vaccines with about 80% protective efficacy.

2.6 HERPES SIMPLEX VIRUS

Herpes simplex virus (HSV) is a member of the Herpitoviridae family, andis distributed worldwide. Transmission of the virus occurs via directcontact from person-to-person, with common entry of HSV-1 through theoral cavity and infection of HSV-2 through the genital tract. Theprevalence of HSV antibody is inversely proportional to socioeconomicstatus, with close to 100% of HSV-positive adults in underdevelopedcountries and 30-50% in developed countries. Acyclovir, idoxuridine (IDUor 2-deoxy-5-iodouridine), trifluridine and vidarabine (adeninearabinoside, Ara-A) are effective in treatment of various HSVinfections, with trifluridine and acyclovir the drugs of choice.Vidarabine, the choice for ophthalmic infections, is not effectivetopically against herpes labialis or other skin/genital infections.Acyclovir is not effective topically when used against recurrent genitalinfections. However, it is effective when used i.v. and p.o. Acycloviris not recommended for routine use in treatment of recurring HSV genitalinfections as ganglionic latency is still present and outbreaks occurafter treatment is stopped.

3. SUMMARY OF THE INVENTION

The present invention relates to a method of treating respiratory virusinfections, comprising administering to a warm-blooded animal, atherapeutically effective amount of an antiviral agent comprising aproanthocyanidin polymer. The proanthocyanidin polymer preferablycontains 2 to 30 flavonoid units, more preferably 2 to 15 flavonoidunits, and most preferably 2 to 11 flavonoid units. The flavonoid unitsinclude but are not limited to catechins, epicatechins, gallocatechins,galloepicatechins, flavanols, flavonols, flavandiols, leucocyanidins,anthocyanidins, or combinations thereof. The flavonoid units can besingly or doubly linked to each other. More specifically, the method ofthe invention can be used to treat respiratory infections induced by arespiratory syncytial virus as well as Parainfluenza virus 3, InfluenzaA, Influenza B virus and virus associated with otitis media and otitisexterna. The proanthocyanidin polymer can be administered intravenously,intraperitoneally, subcutaneously, intramuscularly, orally, topically,or by inhalation.

The proanthocyanidin polymers are effective against most of the virusesimplicated in otitis media. They are soluble in aqueous solutions, arereadily formulated for parenteral, oral, topical (i.e., into ear andnose) administration and thus are admirably suited for use in theprophylaxis or treatment of otitis media.

The proanthocyanidin polymers are similarly effective against theviruses implicated in otitis externa. Because such viruses are locatedoutside of the tympanic membrane, the proanthocyanidin polymers can betopically administered into the ear in a suitable formulation so as tocome directly into contact with the viruses.

The proanthocyanidins useful for treating respiratory virus infectionshave a structure selected from I, II, and III, and their esters, orethers or the corresponding oxonium salts ##STR3## where a, b=1 to 3,x=0 or 1, n=0 to 28, preferably 0 to 13, more preferably 0 to 9.

The proanthocyanidin polymer useful for treating a respiratory virusinfection can comprise 2 to 30, preferably 2 to 15, most preferably 2 to11 monomeric flavanoid units having structure IV, or esters, ethers orcorresponding oxonium salts thereof. ##STR4## where x, y=1 to 3, z=1 or2.

Another embodiment of the invention further relates to a method oftreating virus infections comprising administering, to a warm-bloodedanimal, a therapeutically effective amount of antiviral agent comprisinga proanthocyanidin polymer containing 6 to 0 flavonoid units, preferably6 to 15 flavonoid units and more preferably 6 to 11 flavonoid units. Theflavonoid units include but are not limited to catechins, epicatechins,gallocatechins, galloepicatechins, flavanols, flavonols, flavandiols,leucocyanidins, anthocyanidins, or combinations thereof. The flavonoidunits can be singly or double linked to each other. The method can beused to treat virus infections which are caused by paramyxovaridae,orthomyxovaridae or herpes viruses. The proanthocyanidin polymer can beadministered intravenously, intraperitoneally, subcutaneously,intramuscularly, orally, topically, or by inhalation.

The present invention also relates to proanthocyanidins useful fortreating virus infections in general having a structure selected from I,II, and III, above, and esters, ethers and corresponding oxonium saltsthereof, where n=4 to 28, preferably 4 to 13, most preferably 4 to 9.

The present invention also relates to novel proanthocyanidins which areobtained from a Croton species and from a Calyphyllum inophylum speciesand their esters, ether and oxonium derivatives. Such proanthocyanidincan be isolated from the whole plant, the bark, the leaves, the roots orthe latex. In a preferred embodiment, the proanthocyanidin can beobtained from Croton lechleri and from Calophyllum inophylum. Thesenovel proanthocyanidin polymers are characterized by IR, UV-visible,and/or ¹³ C NMR spectroscopy.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 Fourier-transform infra-red spectrum of proanthocyanidin polymerA isolated from the Croton tree.

FIG. 2 ¹ H Nuclear magnetic resonance spectrum of proanthocyanidinpolymer A isolated from the Croton tree. Sample was in D₂ O at 400 MHz.

FIG. 3 Broad-band decoupled ¹³ C nuclear magnetic resonance spectrum ofproanthocyanidin polymer A in D₂ O at 100 MHz.

FIG. 4 The ¹³ C NMR spectra of proanthocyanidin polymer B isolated fromCalophyllum inophylum, obtained at 100 MHz in D₂ O.

FIG. 5 Fourier-transform infra-red spectrum of proanthocyanidin polymerB isolated from Calophyllum inoplylum.

FIG. 6 The effect of proanthocyanidin polymer A and Amantadine on lungconsolidation in mice infected with influenza A (HIN1) virus (earlytreatment initiation).

FIG. 7 The effect of proanthocyanidin polymer A and Amantadine on lungvirus titers in mice infected with influenza A (HIN1) virus (earlytreatment initiation).

FIG. 8 The effect of proanthocyanidin polymer A and Amantadine onarterial oxygen saturation (SaO₂) in male mice infected with influenza A(HIN1) virus (early initiation).

FIG. 9 The effect of proanthocyanidin polymer A and Amantadine onarterial oxygen saturation (SaO₂) in female mice infected with influenzaA (HIN1) virus (early treatment initiation).

FIG. 10 The effect of proanthocyanidin polymer A and Amantadine on lungconsolidation in mice infected with influenza A (HIN1) virus (latetreatment initiation).

FIG. 11 The effect of proanthocyanidin polymer A on lung titers in miceinfected with influenza A (HIN1) virus (late treatment initiation).

FIG. 12 The effect of proanthocyanidin polymer A and Amantadine onarterial oxygen saturation (SaO₂) in male mice infected with influenza A(HIN1) virus (late treatment initiation).

FIG. 13 The effect of proanthocyanidin polymer A and Amantadine onarterial oxygen saturation (SaO₂) in female mice infected with influenzaA (HIN1) virus (late treatment initiation).

FIG. 14 The effect of Ribavirin on lung virus titers in mice infectedwith influenza A (HIN1) virus.

FIG. 15 The effect of intraperitoneal Ribavirin treatment on lungconsolidation in influenza A (HIN1) virus infected mice.

FIG. 16 The effect of intraperitoneal Ribavirin treatment on blood SaO₂% in influenza A (HIN1) virus infected mice.

FIG. 17 The effect of proanthocyanidin polymer A and Ganciclovir onHSV-2 vaginal lesions in mice.

FIG. 18 The effect of proanthocyanidin polymer A in 5% and 10% topicalformulations, compared to placebo and acyclovir, in a 5% topicalformulation.

5. DETAILED DESCRIPTION OF THE INVENTION 5.1 USEFUL PROANTHOCYANIDINPOLYMERS

Proanthocyanidin oligomers or polymers, useful for the presentanti-viral methods are comprised of monomeric units ofleucoanthocyanidins. Leucoanthocyanidins are generally monomericflavonoids which include catechins, epicatechins, gallocatechins,galloepicatechins, flavanols, flavonols, and flavan-3,4-diols,leucocyanidins and anthocyanidins. The proanthocyanidin polymers usefulfor treating respiratory virus infections have 2 to 30 flavonoid units,preferably 2 to 15 flavonoid units, and most preferably 2 to 11flavonoid units. The proanthocyanidin polymers useful for treating virusinfections in general, including but not limited to, infections relatedto influenza viruses, parainfluenza viruses, viruses of the types knownas paramyxoviridae, orthomyxovaridae, and herpes viruses, have 6 to 30flavonoid units, preferably 2 to 15 flavonoid units, and most preferably6 to 11 flavonoid units

Proanthocyanidin polymers having a varying number of flavonoid units areknown and have been reported, for example, in W. L. Mattice, et al.Phytochemistry, 23, p. 1309-1311 (1984); Z. Czochanska, et al. J.C.S.Chem. Comm., 375 (1979); W. T. Jones, et al. Photochemstry, 15. p.1407-1409 (1976); E. Haslam Plant Polvohenols. p. 75 (1989), which areall incorporated by reference. Those polymers havings the recited rangesof flavonoids units and described in these references are useful for themethods of the present invention.

5.2 METHODS FOR OBTAINING USEFUL PROANTHOCYANIDIN POLYMERS 5.2.1ISOLATION

Useful proanthocyanidin polymers are obtained from various plantsincluding but not limited to the classes Filices, Coniferae,Monocotyledoneae, and Dicotyledonae (J. B. Harborne, THE FLAVONOIDS,ADVANCES IN RESEARCH SINCE 1980 (1988)). They can be obtained using theentire tree or plant, the bark, stems, roots or latex.

These plant source materials are extracted with water and/or a watermiscible solvent. The preferred solvents are alcohol of 1-3 carbon atomsor acetone. The aqueous extract is used directly or after solventremoval. If the solvent is removed, the solid residue is redissolved ina solvent, preferably a lower alcohol or acetone, and insolublematerials are discarded. The soluble fraction is subjected to gelfiltration (e.g., over a Sephadex), reversed-phase column chromatography(e.g., C-8), or gel-permeation chromatography (e.g., divinyl benzenecross-linked gels) such as PL-GEL or membranes (e.g., an Amiconmembrane) using water or water and a water miscible solvent, with orwithout a buffer, as the mobile phase. The water miscible solvent ispreferably a 1-3 carbon alcohol, acetone or acetonitrile.

The useful proanthocyanidin polymers of this invention are the fractionsdetected by ultraviolet (uv) They have their major UV absorption maximabetween about 200-350 nm, with peaks usually at 200-210 nm, and 275-285nm.

With regard to the novel proanthocyanidin polymer compositions whichform part of the basis of this invention, such polymers can be obtainedfrom the Croton tree or Calophyllum species by using the above-describedextraction method. The soluble fraction is subjected to gel filtration,reversed-phase column chromatography, gel-permeation chromatography, ormembranes. Using water or water and a water miscible solvent, with orwithout a buffer as the mobile phase and the relevant fractionscontaining the polymers are detected using uv at a range of about200-350 nm.

According to a preferred embodiment of the present invention, a novelproanthocyanidin polymer can be prepared as follows: Latex obtained froma Croton tree (e.g., Croton lechleri) or from Calophyllum inophylum isused directly or is concentrated by the removal of water (e.g.,lyophilized). It is extracted with water, a lower alcohol of about 1-3carbons, or with acetone to give an aqueous soluble fraction. Theaqueous-soluble fraction of the lyophilized or otherwise concentratedmaterial is subjected to partitioning by ethyl acetate/water solutionand to gel filtration (e.g., Sephadex) using water and/or water andalcohol and/or water and acetone, with or without a buffer as the mobilephase. The fractions containing a chromophoric band are detected byultraviolet (uv) (λmax about 200-350 nm) and collected and concentratedand subjected to gel filtration again, if necessary, to yield theproanthocyanidin polymer. Alternatively, the aqueous-soluble fractiondescribed above is lyophilized or concentrated or is used directly andis subjected to reversed-phase column chromatography (RP HPLC), usingwater and/or water and acetone and/or water and alcohol as the mobilephase. The fractions containing a chromophoric band detected y uv (maxabout 200-350 nm) are collected and concentrated, and subjected to RPHPLC again, if necessary, to yield the proanthocyanidin polymer.Alternatively, the aqueous-soluble fraction, described above, islyophilized, concentrated or is directly subjected to gel permeationchromatography (GPC), using water and/or water and alcohol and/or waterand acetonitrile, with or without a buffer, as the mobile phase. Thefractions containing a chromophoric band detected by uv at λmax about195-350 nm are collected and concentrated and subjected to GPC again, ifnecessary, to yield the proanthocyanidin polymer. See examples inSections 6.1-6.3, infra.

As demonstrated in section 6.2 when chromatographed on a Perkin ElmerLC-620 using Polymer Labs PL-gel 5m 500 A, 300×7.5 mm and THF-water(95:5 v/v) system at a flow rate of 1 ml/min, proanthocyanidin A has aretention time of 7.2 min. and proanthocyanidin B has a retention timeof 6.5 min. Typical spectra for proanthocyanidin polymers are shown inFIGS. 1 and 5.

5.2.2 SYNTHESIS OF USEFUL PROANTHOCYANIDIN POLYMERS AND DERIVATIVESTHEREOF

It is known that leucoanthocyanidins can be condensed in mildly alkalineor acidic solutions to form proanthocyanidins. As an example, L. Y. Fooand R. W. Hemingway, J. Chem. Soc., Chem. Commun., 85 (1984) were ableto synthesize a trimer from flavanol monomers. J. A. Delcoun, et al., J.Chem. Soc. Perkin Trans. I., 1711 (1983) teaches that under mild, lowpH, conditions and an excess amount of (+)-catechin, condensationoccurred to form higher and predominantly linear [4,8]-linked oligomers.Specifically, dimers, trimers and tetramers of the flavonoid analogueswere synthesized and characterized by NMR spectroscopy. By use of suchcondensation reactions proanthocyanidin polymers of up to about 30flavonoid units can be synthesized for use in the methods of theinvention. As an alternative to the above synthesis, enzymatic synthesiscan be employed to produce the useful proanthocyanidin polymers. Thenovel proanthocyanidin polymer compositions of the present inventionspecifically exemplified herein can also be synthesized by suchreactions.

Esters of proanthocyanidin polymers can be prepared by standard methodsof acylation such as by the reaction of the polymer with acid chloridesor acid anhydrides in the presence of base. Of particular utility is thepreparation by reaction with acid anhydrides in pyridine as utilized forthe acylation of certain other lower molecular weight flavonoids byThompson, et al., JCS, 1387 (1972), Fletcher, et al., JCS, 1628 (1977)and Hemingway, JCS, 1299 (1982).

Ethers can be prepared by standard methods of etherification such as byreaction of the polymer with alkyl halides and alkyl tosylates in bases.Methyl ethers are readily prepared by the use of diazomethane asutilized in the etherification of certain other low molecular weightflavonoids by Thompson, JCS, 1387 (1972), Hemingway, JCS, 1299 (1982).

The oxonium salts (sometimes referred to as pyrilium salts oranthocyanidins) may be prepared by the so-called Bate-Smith reaction(Chemistry and Industry, 1953, 377), by warming with aqueous oralcoholic acids (see also, Thompson, et al., JCS, 1387 (1972) andreferences cited in Haslam, PLANT POLYPHENOLS, 1989, p. 28, CambridgePress). This reaction can be advantageously catalyzed by iron (Porter etal., Phytochemistry, 25, 223 (1986)).

5.3 CHARACTERIZATION OF NOVEL PROANTHOCYANIDIN POLYMERS

Novel proanthocyanidin polymers prepared according to the presentinvention have solubility in methanol and water. The polymers aresoluble in water and aqueous solution including alcohol solutions to adegree of at least about 10 mg/ml.

The proanthocyanidin polymers have been analyzed by a number of methodsto determine their molecular weight and various other chemical andphysical features. Various stereoisomers of proanthocyanidin polymershave been obtained and are within the scope of the present invention.

Column chromatography has been used to isolate water soluble fractionscontaining novel proanthocyanidin polymers varying in average molecularweight from about 700 daltons to about 3000 daltons, which correspondsto 2-3 to 9-11 average flavonoid units, respectively.

¹³ C-NMR spectroscopy indicates that the proanthocyanidin polymerscontain flavonoid moieties in which the individual flavonoid ring unitspossess various stereochemistries.

UV-visible spectroscopy is consistent with the possible presence of aflavylium moiety or moieties within certain of the proanthocyanidinpolymers.

The characterization of proanthocyanidin polymer A and proanthocyanidinpolymer B is further discussed in the examples at Sections 6.3 and 6.5.Functional derivatives of these polymers can be made and may be usefulas intermediate for the preparation of useful proanthocyanidin polymers.

5.4 PROPHYLACTIC AND THERAPEUTIC USES OF PROANTHOCYANIDIN POLYMERS

Proanthocyanidin polymers have been shown to be active in vitro and invivo against a wide variety of viruses, and can be advantageously usedin prophylactic and therapeutic applications against diseases induced bysuch viruses.

The proanthocyanidin polymers can be used either alone or in combinationwith other antiviral or antimicrobial agents to prevent and/or treatdiseases induced by or complicated with viral infections from virusesincluding, but not limited to: paramyxovaridae such as respiratorysyncytial virus, orthomyxovaridae such as influenza A, B and C, andherpes viruses such as Herpes Simplex virus.

Proanthocyanidin polymers have various advantages in the treatment ofviral infections including, but not limited to:

1) a broad range of antiviral activity;

2) very low toxicity;

3) no teratogenicity; and

4) application to both systemic as well as localized (i.e., topical)applications.

5.5 ROUTES OF ADMINISTRATION

The proanthocyanidin polymers of the present invention can beadministered for prophylactic and therapeutic applications by a numberof routes, including but not limited to: oral, injection including butnot limited to, intravenous, intraperitoneal, subcutaneous,intramuscular, etc., by topical application such as to nasal,nasopharyngeal linings and into the ear, and by inhalation viaaerosolization and application to respiratory tract linings, etc.

When used according to the present invention against viruses, effectivedose ranges of the proanthocyanidin polymers are about 5.0-about 30mg/kg if given orally (P.O.), about 0.1-about 10 mg/kg if givenintraperitoneally (I.P.), and about 5-about 30 mg/kg/day if given byaerosol. If given topically, the proanthocyanidin polymer is applied ina suitable vehicle at a concentration of about 5 to about 15%.

When administered to warm-blooded animals, including humans, theproanthocyanidins may be combined with water, an aqueous solution or anyphysiologically acceptable carrier or vehicle.

The following series of Examples are presented for purposes ofillustration and not by way of limitation on the scope of the invention.

6. EXAMPLES 6.1 ISOLATION OF A NOVEL PROANTHOCYANIDIN POLYMER FROM ACROTON LECHLERI SPECIES (PROANTHOCYANIDIN POLYMER A

In one series of experiments, a novel proanthocyanidin polymer wasobtained as follows:

C. lechleri trees were tapped and felled near the village of San Pablode Cuyana on the Nanay River 100 kilometers from Iquitos, Peru. Thelatex was obtained over a period of 24 hours by scoring the trees.

The latex (11) obtained from the Croton lechleri trees was diluted withisopropanol in the ratio of 1 part latex to 3 parts isopropanol (31) andallowed to stand for 15 hours at 10° C. The resultantisopropanol-diluted latex was centrifuged, and the insoluble materialwas removed by decantation, leaving the mother liquor (3.7 l). Themother liquor was concentrated to dryness by rotoevaporation at 33° C.to give 240 g of a deep-red brown powder. This concentrated material(980 g) was subjected to gel filtration using Sephadex CM-50 with water(20 l) as the mobile phase. The early-eluting fractions containing a redpigment were detected at λ200-350 nm or by visual detection of a redband eluting through a transparent column. To isolate theproanthocyanidin polymer, the early-eluting fractions were collected andsubjected to gel filtration chromatography and/or HPLC as describedbelow.

The HPLC isolation was performed by injecting 5 μl of concentratedsamples onto a Perkin-Elmer LC Analyst liquid chromatograph systemequipped with an IC-200 autosampler, LC 600 pumps and a LC-235 diodearray detector using a Waters Ultrahydrogel 500 GPC column with Burdickand Jackson HPLC-grade water at 0.8 ml/min at ambient temperature, withdetection at λ280 and 195 nm. The proanthocyanidin polymer Was found tohave a retention time of 4.6-5.6 minutes depending on sampleconcentration, column conditioning and temperature.

The red-pigment containing fractions isolated from Sephadex CM-50 werecombined and subjected to gel filtration chromatography over ToyopearlHW-40S (2.5 L), using water, 10% acetone/water, 20% acetone/water, andfinally 40% acetone/water in a step gradient fashion. The late-elutingred fractions detected by uv-vis as λmax 340 nm, were combined andconcentrated for further purification on HPLC as described above. Thecombined fractions isolated from HPLC were again subjected to gelfiltration chromatography over Toyopearl HW-40S to yield aproanthocyanidin polymer designated proanthocyanidin polymer A.

In another series of experiments, a novel proanthocyanidin polymer wasobtained as follows:

Four liters of cold crude latex from the C. lechleri (or from the entiremacerated plant, the bark or the roots) were diluted with twelve litersof isopropanol, stirred and stored at 5° C. for 15 hours. The residuewas removed by filtration and the solution evaporated to dryness, invacuo, yielding about 970 g. of solids.

The solids were added, with stirring, to 6 l of water and 3.6 l ofn-butanol. The aqueous phase was separated and concentrated to drynessgiving 700 g of material which was added, with stirring, to 2 lmethanol, and then 12 l of ethyl acetate was added. The solution waskept at 15° for 15 hrs and the solid material removed by filtration anddiscarded. The solution was concentrated to dryness yieldingapproximately 390 g. of crude proanthocyanidin polymer A.

The proanthocyanidin polymer A fraction was concentrated by acombination of cation exchange, adsorption and size exclusionchromatography as follows: 450-600 g of the crude proanthocyanidinpolymer A was passed through a CM-Sephadex C-50 pre-column usingde-ionized water as the eluent. The orange and dark red fractions werecollected and then chromatographed, using de-ionized water, over anotherSephadex C-50 column equipped with a UV detector set at 460 nm, and 1-21 fractions were collected. The initial pink band was discarded and thesubsequent effluent passed through a column containing Sephadex G-50until the broad red-brown band was eluted. 15% aqueous acetone waspassed through the G-50 column until the effluent was colorless.Fractions of the effluent were examined by HPLC as described in Section6.2, and those fractions containing proanthocyanidin polymer A werecombined and evaporated to dryness yielding about 210 g. ofproanthocyanidin polymer A.

6.2 PURIFICATION OF PROANTHOCYANIDIN POLYMER A

About 150 g. of crude proanthocyanidin polymer A, obtained as describedin Section 6.1 above, was dissolved in 300 ml 20% aqueous acetone andwas chromatographed over a mixed-mode gel-permeation/absorption column(Toyopearl HW40S, a spherical methyl methacrylate polymer of 40 μmparticle size). 16 l were collected, then the eluting solvent waschanged to 40% aqueous acetone and 8 more liters were collected, thenthe elution was continued with 4 l of 60% aqueous acetone. The fractionscontaining proanthocyanidin polymer A, as shown by HPLC (See Section6.3), were combined and the solvent removed in vacuo yieldingapproximately 57 g. of solid.

Final purification was accomplished by a combination of adsorption andsize-exclusion chromatography: 50-75 g. of the above solid weredissolved in 90% ethanol and introduced onto a column containingSephadex LH-20 (a cross-linked dextran gel with hydroxypropyl groupsattached by ether linkages to glucose units of the dextran chains).Elution was carried out using 10 1 of 90% aqueous ethanol, then 15 l of20% aqueous acetone, 5 l of 40% aqueous acetone, 5 l of 50% aqueousacetone and then 5 l of 60% aqueous acetone. Fractions were collected at2 l intervals and assayed by HPLC. The fractions containingproanthocyanidin polymer A were combined and evaporated to dryness invacuo at 35° giving 35 g. of pure proanthocyanidin polymer A.

For the HPLC, a 30 cm gel-permeation column for non-aqueous mobilephases was used in which the stationary phase is Polymer LaboratoriesPL-Gel 5m 500Å (a divinylbenzene-polystyrene polymer, 5μ particles, 500Åpore size). The HPLC system was equipped with a diode array detectorthat generates UV spectra, with the detector set at 280 nm.

Samples are dissolved in 95% aq THF, which is also the developingsolvent. At a flow rate of ml/min, the proanthocyanidin polymer A peakmaximum has a retention time of 7.2±0.5 min.; proanthocyanidin polymer Bhas a retention time of 6.5±0.5 min, indicating that proanthocyanidinpolymer B has a larger molecular weight than proanthocyanidin polymer A.

6.3 CHEMICAL IDENTIFICATION AND STRUCTURAL FEATURES OF THEPROANTHOCYANIDIN POLYMER A

In the infrared, the proanthocyanidin polymer A of the invention,obtained as described in Section 6.1 above, shows a very broad intensepeak ranging from 3550-2500 and other peaks at 1612, 1449, 1348, 1202,1144, 1107, 1068 and 1027 cm-1. See FIG. 1.

The 1H NMR spectrum in D₂ O (400 MHz 24° C.) exhibited very broad peaksat δ7.1, 6 9, 6.1, 4.7 and 2.8 ppm. See FIG. 2.

Ultraviolet-visible spectral analysis in H₂ O revealed broad peaks atλ202, 235, (shoulder), 275, 305 (shoulder), 460 and trailing greaterthan 600 nm. Although the UV data of the present polymer closelyresembles those of other known proanthocyanidins, the visible data areclearly different. The known proanthocyanidin monomers and polymers arecolorless (λ205, 240, 275 nm) and have no absorption in the visiblerange, whereas certain of the isolated novel proanthocyanidin polymer Aof the present invention are colored and have a visible absorption at460 nm. The color of the proanthocyanidin polymer suggests the presenceof a flavylium moiety or moieties within the proanthocyanidin polymer.This is consistent with the visible spectroscopic data reported forclosely related monomeric anthocyanins which contain the flavyliummoiety (λ460-560 nm).

The 13C NMR spectra of the proanthocyanidin polymer was obtained at 100MHz in D₂ O. The experiments performed were broad-band decoupled. Asshown in FIG. 3, the 13C NMR spectrum in D₂ O exhibited very broad peaksat δ155(C-5, C-7, C-9), 145(C-3', C-5')*, 143(C-3', C-4'), 130(C-1',C'-1'*, C-4'*), 128(C-1'), 121(C-6), 116(C-2', C-5'), 109(C-8, C-2',C-6'*), 97(C-6), 82(C-2), 76(C-2), 73(C-3), and 38(C-4) ppm, for the*prodelphinidin B ring. The 13C NMR data suggest some key points ofstructural differences between the novel antiviral proanthocyanidinpolymers of the present invention and known proanthocyanidin polymers.The most significant difference is a substantially larger peak in theregion of 109 ppm for the proanthocyanidin polymers of this inventionwhen compared with 13C NMR spectra of other proanthocyanidin polymersknown in the literature (compare with spectra in Czochanska et al.,1979, J.C.S. Chem. Comm., p. 375-77; in Harborne, J. B. and Mabry, T. J.ed., The Flavonoids: Advances in Research, Chapman and Hall, N.Y., 1982,pp. 51-132, incorporated herein by reference). The 13C NMR data of thepolymer is indicative of the proanthocyanidin class of polymers. Inparticular, the 13C NMR chemical shift data of the isolatedproanthocyanidin polymer (C-6'=132 ppm, C-2'=115 pm, C-3' and C- 4'=145ppm, C-5'=116 ppm, C-6'-107 ppm, C-3' and C-5'=146 ppm, C-4'-133 ppm) isconsistent with a polymer composed of procyanidin B ring moieties withthe individual flavonol C ring units possessing both the 2,3-trans and3,4-trans [similar to (+)-catechin; C-2=83 ppm, C-3=73 ppm, C-4=38ppm]and 2,3-cis-3,4-trans [similar to (-)-epicatechin; C-2=77 ppm, C-3=73ppm, C-4=73 ppm] stereochemistries. The present experiment dataindicates that proanthocyanidin polymer A is comprised of catechin,epicatechin, gallocatechin and galloepicatechin. The HPLC data suggestthat the average number of flavonoid units is about 7. The HPLC dataalso suggests that number of flavonoid units vary from 2 to 11.

The proanthocyanidin polymer A is soluble in methanol, water and aqueoussolutions. The proanthocyanidin polymer A is soluble in water atconcentrations of at least about 10 mg/ml. The polymer has lowersolubility in normal saline and other salt solutions. Mass spectralanalysis indicate that proanthocyanidin polymer A has a molecular weightaverage of about 2,100 daltons.

6.4 ISOLATION OF A PROANTHOCYANIDIN POLYMER FROM CALOPHYLLUM INOPHYLUM(PROANTHOCYANIDIN POLYMER B)

A novel proanthocyanidin polymer according to the present invention canbe isolated by a method similar to that described above in Section 6.1from the entire macerated plant, the bark, the leaves, the roots or thelatex of Calophyllum inophylum. AccorDing to a preferred method, theproanthocyanidin polymer is obtained by the method described in Section6.1, except that water is used as the preferred extraction solvent.

In one series of experiments, a novel proanthocyanidin polymer,designated proanthocyanidin polymer B, was obtained from Calophylluminophylum latex.

2,849 g of the latex from Calophyllum inophylum was mixed with 12.4 l ofa 1:1 mixture of isopropanol and water, stirred and stored at roomtemperature for 36 hours. The residue was removed by filtration and thesolution was evaporated to dryness, in vacuo, giving 133.5 g of solids.

The solids were added, with stirring, to 30 g of methanol. The solutionwas then filtered away from the solids, and a 1:1 mixture of water andethyl acetate was added. The water fraction was separated and n-butylalcohol was added. The water fraction was separated from the alcoholfraction and concentrated to dryness yielding approximately 10.4 g ofcrude proanthocyanidin polymer B.

The crude proanthocyanidin polymer B was passed through a CM-50 SephadexCC column using de-ionized water as the eluting solvent. A red band wascollected and fractionated using a LH-20 CC column, eluted with 70%aqueous ethanol solution and 20%, 50% and 70% aqueous acetone solutionsto give proanthocyanidin polymer B. At lower concentrations, a solutionof proanthocyanidin polymer B is essentially colorless. At higherconcentrations, the solution of proanthocyanidin polymer B is tan.

1 6.5 CHEMICAL IDENTIFICATION AND STRUCTURAL FEATURES OF THEPROANTHOCYANIDIN POLYMER B

The ¹³ C NMR spectrum of proanthocyanidin polymer B (FIG. 4) confirmsthat the polymer is a member of the class of proanthocyanidin polymersand comprises principally catechin and epicatechin monomeric flavonoidunits. Gel permeation chromatography (GPC) indicates thatproanthocyanidin polymer B has a molecular weight that is larger thanproanthocyanidin polymer A. Consistent with the GPC data, HPLC-GPCretention time of proanthocyanidin polymer B is shorter than that forproanthocyanidin polymer A under the same conditions--again indicatingthe larger size of proanthocyanidin polymer B. HPLC indicates thatpolymer B has a molecular weight average of about 3000 daltons,corresponding to an average number of flavonoid units of about 10. TheHPLC data also suggests that the number of flavonoid units vary from 5to 16. Mass spectral analysis indicates that proanthocyanidin polymer Ahas a molecular weight average of about 2100 daltons.

The Fourier-transform infra-red spectrum of proanthocyanidin polymer Bis quite similar to that of proanthocyanidin polymer A (See FIG. 5 andFIG. 1).

Likewise, the UV-visible spectrum of proanthocyanidin polymer B issimilar to that of proanthocyanidin polymer A except for the absence ofthe peak at 460 nm.

7. SCREENING OF PROANTHOCYANIDIN POLYMER A FOR ANTIVIRAL ACTIVITY

In one series of experiments, proanthocyanidin polymer A was tested forantiviral activity against the following viruses: Respiratory syncytialvirus subtype variants, A2-Tracey, A-Long, B-46791, B-47063 and B-18537;parainfluenza virus, type 3 (PIV-3); adenoviruses, type 5 and 7;influenza, A-Taiwan (HIN1); A-Leningrad (H3N2); A-Japan; A-PortChalmbers; A-NWS33; B-USSR; B-Tama; B-RF; measles virus, and Edmonstonstrain.

All viruses were obtained from the Influenza Research Center, BaylorCollege of Medicine, Houston, Texas, with the exception of measles viruswhich was obtained from ATCC. For comparison, ribavirin was included inthe screening assay.

The following procedure was used to assay for antiviral activity. Assayswere performed in 96-well tissue culture plates. All dilutions andtissue culture suspensions were prepared in minimal essential mediumcontaining antibiotics penicillin and streptomycin and 2% fetal calfserum (2% FCS-MEM). Test compounds (0.05 ml) were added in quadruplicateto wells of the test plates containing a subconfluent monolayer of HEp2cells (ca. 3×10³ cells). The compounds were diluted using serial 2-folddilutions usually starting with a final concentration of 1 mg/ml.Approximately 100 median tissue culture infectious doses (TCID₅₀) of theappropriate test virus in 0.05 ml was added. Tissue control wellscontained medium, without virus or antiviral compound, and antiviralcontrol wells contained antiviral compound without virus. Ribavirin wasincluded in each assay as a positive antiviral control, except for theadenoviruses where Ribavirin fails to demonstrate antiviral activity.Back titrations of each test virus were also included in each assay. Allplates were incubated at 37° C. in a 5% CO₂ incubator. Virus controlwells were observed daily. When these wells exhibited 80-100% cytopathiceffect (CPE), all wells were observed for CPE. In addition to visual andmicroscopic observance of CPE, inhibition of syncytial formation wasused to confirm activity against RSV.

In each antiviral assay, a 50% minimal inhibitory concentration (ED₅₀)was determined. The ED₅₀ was calculated by determining the medianminimal concentration of compound tested in wells inhibiting CPE 50%compared to virus control wells. The actual calculation of the ED₅₀value was done with the aid of the computer program, "Dose-effectanalysis with microcomputers" of Chou et al., 1984, Adv. Enz. Regul.,22:27-55.

The effect of each compound on the growth of uninfected tissue culturecells, seeded at low densities to allow rapid growth, was alsoevaluated. From this assay, a 50% minimal toxic concentration (ID₅₀) wasdetermined. The assay for cell toxicity or inhibition of cell growthinvolved the following procedure. Test compounds (0.1 ml/well) wereserially diluted 2-ofld. To the appropriate wells were added 0.1 ml ofhuman HeLa, A549 or HEp2 cells, 0.1 ml of mouse L929 cells, 0.1 m ofmonkey Vero cells or 0.1 ml of canine MDCK cells. Approximately 3×10³cells were added to each well. Control wells consisted of wellscontaining a range of cell concentration (e.g., 3×10³ cells, 1:2dilution of this number, 1:4, 1:8 and 1:16) in medium without anyantiviral compound. A vehicle control consisting of serial dilutions ofwhatever vehicle was used for a particular compound (e.g., 10% DMSO, 50%methanol) was also included in each assay in duplicate. All plates wereincubated at 37° C. in a 5% CO₂ incubator. After control wellscontaining cells, but no test compound, reached confluency,3-[4,5-Dimethylthiazol-2-yl]-2,5-Diphenyltetrazolium Bromide (MTT) wasadded to all wells. Three hours later, acid alcohol (0.1 ml of 0.4N HClin isopropyl alcohol) was added to each well to solubilize anyprecipitate formed in each well. Plates were read on a plate reader (UVMAX, Molecular Devices) to determine the optical densities in each well.The median concentration of antiviral compound in the last wells causinga 50 % reduction in O.D. was determined and termed "ID₅₀ ". All wells inthe assay were also observed microscopically for inhibition of cellgrowth. The results are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        Effect of In Vitro Screening for Antiviral Activity                           with Proanthocyanidin Polymer A                                               ED.sub.50 (μg/ml)                                                          Virus        Proanthocyanidin Polymer A                                                                       Ribavirin                                     ______________________________________                                        Experiment 1                                                                  RSV              8.5               4.0                                        Parainfluenza virus 3                                                                       >94                 15.6                                        Influenza A    88                 15.0                                        Influenza B   125                 18.0                                        Adenovirus   >125               --                                            Rhinovirus   >125                 125                                         Measles      >125                 31.3                                        Experiment 2                                                                  RSV              17.2             24.2                                        Parainfluenza virus                                                                            38.3             15.7                                        Influenza A      31.3             14.3                                        Influenza B   125                 18.0                                        Adenovirus 5 >250               >1000                                         Adenovirus 7 >250               >1000                                         Rhinovirus   >125                 125                                         Measles      >125                 31.3                                        Experiment 3                                                                  RSV            12                 31                                          Parainfluenza virus 3                                                                        5                  12                                          Influenza A    4                  14                                          Adenovirus 7 >250               >1000                                         ______________________________________                                    

In the first experiment of Table 1, the ED₅₀ for the proanthocyanidinpolymer A was determined to be 8.5 μg/ml for RSV. This activity comparesquite closely to ribavirin with a ED₅₀ determined to be 4.0 μg/ml forRSV. The ID₅₀ (an index of cell toxicity) of the proanthocyanidinpolymer A was 94 μg/ml. In the second experiment of Table 1, the ED₅₀for the proanthocyanidin polymer A was 17.2 μg/ml for RSV as compared to24.2 for ribavirin, and the ID₅₀ was 250 μg/ml. In the third experimentof Table 1 the ED₅₀ for the proanthocyanidin polymer A was 12 μg/ml andthe ID₅₀ was greater than 250 μg/ml.

The results of antiviral activity of the proanthocyanidin polymer Acompared to ribavirin in each assay was determined by calculating thedrug Selective Index (SI), defined as the ratio of ID₅₀ /ED₅₀. TheSelective Index of the proanthocyanidin polymer A for RSV was 11 and14.5 and 21 in the three experiments.

Virus rating (VR) was obtained by averaging the sum of the CPE values (0for no CPE; 4 for 100% CPE) assigned to treated, infected monolayers ateach compound concentration. This average is subtracted from the averageof the sum of the CPE values in an equal number of virus control wells.A further adjustment was made to reflect any observed cytotoxicity.Values for VR were assigned as follows: VR greater than 1, defined asdefinite anti-viral activity; a VR of 0.5-0.9, as moderate toquestionable activity; a VR of 0.1-0.5, as slight activity. The slightactivity could be attributable to cytotoxicity or cytopathic effects ofthe compound per se. The VR of the proanthocyanidin polymer A againstRSV was 1.5, indicating definite anti-viral activity in these in vitroassays. Also, the proanthocyanidin polymer A demonstrated substantialanti-viral activity against parainfluenza and influenza A viruses in allthree experiments.

In another series of experiments, polymer A was tested for antiviralactivity against RSV, types A and B, influenza types A (Flu-A) and B(Flu-B), and parainfluenza types (PIV-1) and 3 (PIV-3).

The cell lines used were Human HeLa, A549 and HEp-2 cells, mouse L929cells, monkey Vero cells, canine MDCK cells.

The effect of the proanthocyanidin polymer A on the viability and growthof cells was determined by measurement of mitochondrial respiration andexpressed as a 50% minimal toxic concentration (ID₅₀). In this assay, asin the ED50 assay, the proanthocyanidin polymer A was assayed in serial2-fold dilutions in quadruplicate across 96-well microtitre plates.Again, wells were seeded at the low density of 3×10³ cells per well toallow for rapid growth. Vehicle controls which consisted of serialdilutions of vehicle (which, in the case of proanthocyanidin polymer A,was water) were included in each assay in duplicate. Blanks whichconsisted of media alone and media plus drug were also included. Allplates were incubated at 37° C. in a 5% CO₂ incubator until the tipconcentration of cells in control wells reached confluency, usually 3days. All wells in each assay were observed microscopically forcyctotoxicity. Then 0.05 ml of MTT(3-[4,5-dimethythiazole-2-yl]-2,5-diaphenyltetrazolium bromide) (5 mg/mlin PBS) was added to all wells and the incubation continued. Three hourslater acid alcohol (0.05 ml of N HCl in isopropyl alcohol) was added toeach well. The optical densities (O.D.) of each well were read at 490 nmusing a plate reader (UV MAX, Molecular Devices). The percent viabilityof cells at each concentration of drug was calculated by dividing themean of the O.D.'s of the drug treated wells (minus the mean of the drugblank) by the mean of the O.D.'s of control wells (minus the mean of theblank), and multiplying by 100. The effect of the vehicle on cellviability was similarly calculated. A dose response curve for thetoxicity of proanthocyanidin polymer A was generated and the ID₅₀determined.

The following procedure was used to assay for antiviral activity. Assayswere performed in 96-well tissue culture plates. All dilutions andtissue culture suspensions were prepared in minimal essential mediumcontaining antibiotics penicillin and streptomycin and 2% fetal calfserum (2% FCS-MEM). Test compounds (0.05 ml) were added in quadruplicateto wells of the test plates containing a subconfluent monolayer of HEp2cells (ca. 3×10³ cells). The compounds were diluted using serial 2-folddilutions usually starting with a final concentration of 1 mg/ml.Approximately 100 median tissue culture infectious doses (TCID₅₀) of theappropriate test virus in 0.05 ml was added. Tissue control wellscontained medium, without virus or antiviral compound, and antiviralcontrol wells contained antiviral compound without virus. Ribavirin wasincluded in each assay as a positive antiviral control, except for theadenoviruses where Ribavirin fails to demonstrate antiviral activity.Back titrations of each test virus were also included in each assay. Allplates were incubated at 37° C. in a 5% CO₂ incubator. Virus controlwells were observed daily. When these wells exhibited 80-100% cytopathiceffect (CPE), all wells were observed for CPE. In addition to visual andmicroscopic observance of CPE, inhibition of syncytia formation was usedto confirm activity against RSV.

In each antiviral assay, a 50% minimal inhibitory concentration (ED₅₀)was determined. The ED₅₀ was calculated by determining the medianminimal concentration of compound tested in wells inhibiting CPE 50%compared to virus control wells. The actual calculation of the ED₅₀value was done with the aid of the computer program, "Dose-effectanalysis with microcomputers" of Chou et al., 1984, Adv. Enz. Regul.,22:27-55.

The selective index for proanthocyanidin polymer A was then calculatedas the ratio of the ID₅₀ over the ED₅₀.

The results are shown in Tables 2-6. The results indicate thatproanthocyanidin polymer A is as effective as ribavirin for inhibitinggrowth of RSV-A and B, PIV-1, PIV-3, FLU-A and FLU-B in vitro. Theselective index values indicate low cytotoxicity for proanthocyanidinpolymer A.

                  TABLE 2                                                         ______________________________________                                        Effect of Proanthocyanidin Polymer A and                                      Ribavirin Against RSV.                                                        PROANTHOCYANIDIN                                                              POLYMER A (μg/ml)                                                                              RIBAVIRIN (μg/ml)                                      EXP #  ED.sub.50                                                                              ID.sub.50                                                                             S.I.  ED.sub.50                                                                            ID.sub.50                                                                           S.I.                               ______________________________________                                        A-Tracy                                                                              9.0       94.0   10.4  4.0    1000.0                                                                              250.0                              A-Tracy                                                                              6.0      750.0   125.0 1.0    1000.0                                                                               83.3                              A-Tracy                                                                              16.0     750.0   46.0  6.0    1000.0                                                                              166.7                              A-Long 8.0      125.0   15.6  8.0    1000.0                                                                              125.0                              A-Long 31.0     250.0    8.1  31.0   1000.0                                                                               32.3                              A-Long 12.0     250.0   20.8  31.0   1000.0                                                                               32.3                              A-Long 25.0      78.0    3.1  3.0     403.0                                                                              134.3                              A-Tracy                                                                              2.0       94.0   47.0  12.0   1000.0                                                                               83.3                              B-46791                                                                              6.0      188.0   31.0  8.0    1000.0                                                                              125.0                              B-47063                                                                              6.0       98.0   16.3  12.0   1000.0                                                                               83.3                              B-18537                                                                              8.0      188.0   23.5  4.0    1000.0                                                                              250.0                              MEAN   11.7     260.5   31.6  11.9    945.7                                                                              124.1                              S.E.M. 2.7       75.3   10.3  4.0     54.3  22.5                              ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        Effect of Proanthocyanidin Polymer A and Ribavirin                            Against FLU-A.                                                                       PROANTHOCYANIDIN                                                              POLYMER A (μg/ml)                                                                        RIBAVIRIN (μg/ml)                                     EXP #    ED.sub.50                                                                             ID.sub.50                                                                             S.I.  ED.sub.50                                                                            ID.sub.50                                                                           S.I.                              ______________________________________                                        A-Taiwan 16.0    750.0    46.9 8.0    1000.0                                                                              125.0                             A-Taiwan 4.0     750.0   187.5 6.0    1000.0                                                                              166.7                             A-Taiwan 12.0    750.0    62.5 23.0   1000.0                                                                               43.5                             A-Taiwan 2.0      63.0    31.5 12.0   1000.0                                                                               83.3                             A-Taiwan 0.8     250.0   312.5 7.8    1000.0                                                                              128.2                             A-Taiwan 4.0      25.0    6.3  7.8    1000.0                                                                              128.2                             A-Shanghai                                                                             0.8     187.0   233.8 6.0    1000.0                                                                               83.3                             A-Leningrad                                                                            4.0     750.0   187.5 6.0    1000.0                                                                              166.7                             B-NWS/33 8.0      41.0    5.1  1.0     100.0                                                                              100.0                             B-Japan  12.0     24.0    2.0  1.6     78.0  48.8                             B-Portchalm                                                                            13.0     35.0    2.7  1.0     68.0  68.0                             MEAN     7.0     263.5    81.5 8.2     749.6                                                                              101.9                             S.E.M.   1.6      96.7    33.2 2.0     129.3                                                                               13.3                             ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        Effect of Proanthocyanidin Polymer A and                                      Ribivarin Against FLU-B.                                                             PROANTHOCYANIDIN                                                              POLYMER A (μg/ml)                                                                        RIBAVIRIN (μg/ml)                                     EXP #    ED.sub.50                                                                             ID.sub.50                                                                             S.I.  ED.sub.50                                                                            ID.sub.50                                                                           S.I.                              ______________________________________                                        B-Yamagata                                                                             3.0     187.0    62.3 6.0    1000.0                                                                              166.7                             B-Yamagata                                                                             1.5     187.0   124.7 8.0    1000.0                                                                              125.0                             B-USSR   62.0    750.0    12.1 4.0    1000.0                                                                              250.0                             B-USSR   0.8     250.0   312.5 7.8    1000.0                                                                              128.2                             B-USSR   3.0     250.0    83.3 7.8    1000.0                                                                              128.2                             B-HongKong                                                                             11.0     35.0    3.2  1.0     80.0  80.0                             MEAN     13.6    276.5    99.7 5.8     846.7                                                                              146.3                             S.E.M.   9.8     100.0    46.4 1.1     153.3                                                                               23.6                             ______________________________________                                    

                  TABLE 5                                                         ______________________________________                                        Effect of Proanthocyanidin Polymer A and                                      Ribavirin Against PIV-1.                                                      PROANTHOCYANIDIN                                                              POLYMER A (μg/ml)                                                                              RIBAVIRIN (μg/ml)                                      EXP #   ED.sub.50                                                                             ID.sub.50                                                                             S.I.  ED.sub.50                                                                            ID.sub.50                                                                           S.I.                               ______________________________________                                        PIV-1   0.8     125.0   166.7 12.0   1000.0                                                                              83.3                               PIV-1   2.0     187.0    93.5 12.0   1000.0                                                                              83.3                               PIV-1   6.0     750.0   125.0  8.0   1000.0                                                                              125.0                              MEAN    2.9     354.0   124.9 10.7   1000.0                                                                              97.2                               S.E.M.  1.6     198.8    18.1  1.3     0.0 13.9                               ______________________________________                                    

                  TABLE 6                                                         ______________________________________                                        Effect of Proanthocyanidin Polymer A and                                      Ribavirin Against PIV-3.                                                      PROANTHOCYANIDIN                                                              POLYMER A (μg/ml)                                                                              RIBAVIRIN (μg/ml)                                      EXP #   ED.sub.50                                                                             ID.sub.50                                                                             S.I.  ED.sub.50                                                                            ID.sub.50                                                                           S.I.                               ______________________________________                                        PIV-3    94.0   124.0    1.3  16.0   1000.0                                                                               62.5                              PIV-3   250.0   750.0    3.0  46.0   1000.0                                                                               21.7                              PIV-3    93.0   125.0    1.3  8.0    1000.0                                                                              125.0                              PIV-3    16.8   250.0   15.6  16.0   1000.0                                                                               62.5                              PIV-3    5.0    250.0   50.0  8.0    1000.0                                                                              125.0                              PIV-3    84.0    78.0    0.9  8.0    1000.0                                                                              125.0                              PIV-3    8.0    188.0   23.5  6.0    1000.0                                                                              166.7                              MEAN     78.6   252.1   13.7  15.4   1000.0                                                                               98.3                              S.E.M.   32.4    86.6    6.9  5.3      0.0  19.0                              ______________________________________                                    

Fractions of proanthocyanidin polymer A have been further purified byHPLC using a GPC column and standards. Specifically, two fractions havebeen isolated: one fraction having a molecular weight average of 700daltons (corresponding to 2 to 3 flavonoid monomeric units) and thesecond fraction having a molecular weight average of 3000 daltons(corresponding to 9 to 11 flavonoid monomeric units). The two fractionsshow essentially the same chemical shifts in the ¹³ C and ¹ H NMRspectra as the unfractionated proanthocyanidin polymer A.

In vitro experiments indicate both fractions are active against RSV. Thefraction having an average of 2 to 3 flavonoid units has an in vitro RSVantiviral activity of ED₅₀ =10 μg/ml, while the second fraction havingan average of 9-11 flanonoid units has an ED₅₀ =40 μg/ml.

8. EFFECTIVENESS OF PROANTHOCYANIDIN

In vivo and in vitro assays indicate that the novel proanthocyanidinpolymer A of the invention is a useful therapeutic agent for thetreatment of RSV infections. The present invention provides a safertherapeutic treatment for RSV infections without the toxic effectsassociated with Ribavirin, the currently approved composition fortreatment of such infections.

Hispid Cotton Rats were innoculated with RSV A2 intranasally on day 1and given an intraperitoneal dose of 0.1-30.0 mg/kg of theproanthocyanidin polymer prepared in distilled water on day 2, 3 and 4.Control animals received an equal volume of distilled water. In oneexperiment, as a comparison, a dose of 10-90 mg/kg of Ribavirin wasused. All animals were killed on day 5. Lungs were removed and washedvia transpleural lavage. The fluid collected was assessed for RSV titerin a microtiter assay using HEp2 cells.

Assays were performed in 96-well tissue culture plates. All dilutionsand tissue culture suspensions were prepared in minimal essential mediumcontaining antibiotics and 5% fetal calf serum (5% FCS-MEM). Lung lavagefluid was added in quadruplicate to wells of the test plates containinga subconfluent monolayer of cells (ca. 3×10³ cells). Approximately 100median tissue culture infectious doses (TCID₅₀) of the appropriate testvirus in 0.05 ml was added. Tissue control wells contained medium, butno virus. All plates were incubated at 37° C. in a 5% CO₂ incubator.Virus control wells were observed daily. When these wells exhibited80-100% CPE, all wells were observed for CPE. In addition to visual CPE,inhibition of syncytia formation is used to confirm activity againstRSV. Lung RSV titer logm was calculated as described by Dubovi et al.,1983; 1984. The results are shown in Tables 7-8.

As shown in Table 7, for each experiment, the proanthocyanidin polymer Ademonstrated a dose-dependent decrease in RSV titer compared tocontrols. This indicates that the proanthocyanidin polymer A haseffective antiviral activity against RSV in vivo. As shown in Table 8,direct comparison of the antiviral effect of the procyanidin polymer Ato ribavirin (intraperitoneal administration) demonstrates that thepolymer composition of the invention is significantly more potent thatribavirin, the drug currently used to treat RSV infections.

Hispid Cotton Rats were inoculated with RSV A2 intranasally on day 1 andgiven an oral dose of 1.0-10.0 mg/kg of the proanthocyanidin polymer ondays 2, 3 and 4. All animals were killed on day 5 and lung tissues werecollected. Lungs were removed and washed via transpleural lavage. Thefluid collected was assessed for RSV titer in a microtiter assay usingHEp2 cells as described above. Results are shown in Table 9.

As shown in Table 9, the proanthocyanidin polymer administered orallysignificantly lowered RSV titers at 10 mg/kg compared to controls. Thisindicates that the proanthocyanidin polymer has effective antiviralactivity when administered orally against RSV and is more active thanribavirin.

                  TABLE 7                                                         ______________________________________                                        Effect of Proanthocyanidin Polymer A Against                                  RSV Infection of Cotton Rats, Intraperitoneal Dosing                          Treatment      Lung                                                           (Mg/Kg)        titer.sub.a   P                                                ______________________________________                                        EXPERIMENT #1                                                                 Control        3.98 ± 0.14                                                                              --                                                              (9549)                                                          0.1           3.78 ± 0.31                                                                              0.08                                                            (6025) 36.9% kill                                               0.3           3.21 ± 0.05                                                                              0.05                                                            (1621) 83.0% kill                                               1.0           3.28 ± 0.65                                                                              0.04                                                            (1905) 80.1% kill                                              EXPERIMENT #2                                                                 Control        4.3 ± 0.8  --                                                              (20,000)                                                        1.0           3.1 ± 0.5  0.08                                                            (1258) 94% kill                                                 3.0           3.4 ± 0.5  0.05                                                            (2,511) 87% kill                                               10.0           3.6 ± 0.3  0.06                                                            (3,981) 80% kill                                               EXPERIMENT #3                                                                 Control        3.98 ± 0.14                                                                              --                                                              (9,549)                                                         3.0           4.00 ± 0.02 n.s.                                                           (10,000) 0% kill                                               10.0           3.88 ± 0.3 n.s.                                                            (7,585) 20.6% kill                                             30.0           3.70 ± 0.28 n.s.                                                           (5,011) 47.5% kill                                             EXPERIMENT #4                                                                 Control        5.22 ± 0.27                                                                              --                                                              (165,958)                                                       0.1           4.87 ± 0.32                                                                              n.s.                                                            (74,131) 55.3% kill                                             0.3           4.25 ± 0.39                                                                               0.014                                                          (17,782) 89.3% kill                                             1.0           4.27 ± 0.5  0.046                                                          (18,620) 88.8% kill                                             3.0           4.41 ± 0.19                                                                               0.012                                                          (25,703) 84.5% kill                                            EXPERIMENT #5                                                                 Control        4.5 ± 0.2  --                                                              (31,622)                                                       10             4.5 ± 0.2  n.s.                                                            (31,622) 0% kill                                               30             4.0 ± 0.3  0.03                                                            (10,000) 68.4% kill                                            90             2.8 ± 0.3  <0.0001                                                         (631) 98% kill                                                 ______________________________________                                         .sub.a Lung titer represents: RSV titer log .sub.10 /g lung (Mean ±        S.D.)                                                                    

                  TABLE 8                                                         ______________________________________                                        Effect of Proanthocyanidin Polymer A and                                      Ribavirin Against RSV Infection of                                            Cotton Rats, Intraperitoneal Dosing                                                     Lung                                                                          titer.sub.a P                                                       ______________________________________                                        Proanthocyanidin Polymer (Mg/Kg)                                              Control     5.22 ± 0.27                                                                              --                                                              (165,958)                                                          0.1        4.87 ± 0.32                                                                              n.s.                                                            (74,131) 55.33% Kill                                               0.3        4.25 ± 0.39                                                                              0.014                                                           (17,782) 89.29% Kill                                               1.0        4.27 ± 0.5 0.046                                                           (18,620) 88.78% Kill                                               3.0        4.41 ± 0.19                                                                              0.012                                                           (25,703) 84.51% Kill                                              Ribavirin (Mg/Kg)                                                             Control     4.5 ± 0.2  --                                                              (31,622)                                                          10          4.5 ± 0.2  n.s.                                                            (31,622) 0% Kill                                                  30          4.0 ± 0.3  0.03                                                            (10,000) 68.4% Kill                                               90          2.8 ± 0.3  <0.0001                                                         (631) 98% Kill                                                    ______________________________________                                         .sub.a Lung titer represents: RSV titer log .sub.10 /g lung (Mean ±        S.D.)                                                                    

                  TABLE 9                                                         ______________________________________                                        Effect of Oral Proanthocyanidin Polymer A                                     Against RSV Infection of Cotton Rats                                          Treatment      Lung                                                           (Mg/Kg)        titer.sub.a   P                                                ______________________________________                                        EXPERIMENT #1                                                                 Control        4.45 ± 0.50                                                                              --                                                              (28,183)                                                        1.0           3.62 ± 0.42                                                                               0.045                                                          (4168) 85.7% kill                                               3.0           4.22 ± 0.23                                                                              n.s.                                                            (16,595) 41.4% kill                                            10.0           3.95 ± 0.48                                                                              n.s.                                                            (8912) 68.4% kill                                              EXPERIMENT #2                                                                 Control        3.86 ± 0.55                                                                              --                                                              (7,244)                                                         1.0           3.60 ± 0.01                                                                              n.s.                                                            (3,981) 45.0% kill                                              3.0           3.71 ± 0.50                                                                              n.s.                                                            (5,129) 29.2% kill                                             10.0           2.90 ± 0.57                                                                              0.06                                                            (794) 89.0% kill                                               EXPERIMENT #3                                                                 Control        3.90 ± 0.3 --                                                              (7,943)                                                         1.0           3.90 ± 0.8 n.s.                                                            (7,943)       0.0% Kill                                         3.0           4.00 ± 0.8 n.s.                                                            (10,000)      0.0% Kill                                        10.0           2.90 ± 0.5 0.01                                                            (794)         92% Kill                                         Ribavirin      3.30 ± 0.5 0.07                                             (40 mg/kg)     (1,995)       80% Kill                                         ______________________________________                                         .sub.a Lung titer represents RSV titer log .sub.10 /g lung (Mean ±         S.D.)                                                                    

An additional series of experiments conducted as described aboveconfirms the in vivo activity of polymer A against RSV.

Proanthocyanidin polymer A displayed a mean ED₅₀ value of 1.52±0.62mg/kg following 3-day i.p. administration, and 3.6±1.66 mg/kg following3-day p.o. administration. See Table 10. The reference antiviral agent,ribavirin, displayed ED₅₀ values of 40±6, and >90 mg/kg following i.p.and p.o. administration, respectively. Proanthocyanidin polymer A at adose of 10 mg/kg p.o. resulted in a 68-92% reduction in lung RSV titersfollowing oral administration, and a 21-80% reduction following i.p.administration. Ribavirin (40 mg/kg) resulted in a >90% reduction inlung titers following oral administration, and a 50% reduction followingi.p. administration.

                  TABLE 10                                                        ______________________________________                                        Effect of Proanthocyanidin Polymer A                                          Against RSV infection of Hispid Cotton Rats:                                               ED.sub.50 (mg/kg)                                                               Proanthocyanidin                                               Exp. #         Polymer A    Ribavirin                                         ______________________________________                                        3-Day Intraperitoneal Dosing                                                  1              1.0           40                                               2              0.3           30                                               3              3.0           50                                               4              0.3                                                            5              3.0                                                            Mean ± S.E.M.                                                                             1.52 ± 0.62                                                                             40± 5.8                                        3-Day Oral Dosing                                                             1              3.0          >90                                               2              1.0          >90                                               3              10.0                                                           4              3.0                                                            5              1.0                                                            Mean ± SEM  3.6 ± 1.66                                                                              >90                                               ______________________________________                                    

In vivo experiments have also been performed to study the effect ofproanthocyanidin polymer A upon course of respiratory syncytial virusinfection in African green monkeys.

Proanthocyanidin polymer A was weighed out in 25 mg aliquots andrefrigerated as the dry powder until use. Glucose was prepared as a 0.5%solution and refrigerated. A 25 mg sample of proanthocyanidin polymer Awas dissolved in 50 ml 0.5% glucose daily and the solution sterilized byfiltration through a 0.22 μm membrane filter. Subsequent dilutions weremade in 0.5% glucose from the 0.5 mg/ml solution at 1:2 and 1:5 to giveconcentrations of 0.25 mg/ml and 0.1 mg/ml respectively.

Groups of three monkeys were dosed with proanthocyanidin polymer A at0.5 mg/kg, 0.25 mg/kg or 0.1 mg/kg by administering the drug at 1 ml perkg of body weight by intravenous injection. The time of administrationof the drug infusion to each monkey was approximately 1 minute. Acontrol group of three monkeys received 1 ml of the 0.5% glucosesolution.

Four hours after the initial treatment with proanthocyanidin polymer A,each of the monkeys was infected with a 10⁻² dilution of stockrespiratory syncytial virus. The titer of the stock virus was 1×10⁵TCID₅₀ /ml. Each monkey received 1 ml of the 10% virus dilution byintratracheal inoculation and 1 ml applied drop-wise to external nares.The total virus dose therefore was 2×10³ TCID₅₀ /monkey. Treatment withproanthocyanidin polymer A was administered again 8 hours laterresulting in daily doses of proanthocyanidin polymer A of 1.0, 0.5, and0.2 mg/kg with three monkeys per group. Twice daily treatment continuedat 8 a.m. and 8 p.m. for a total of seven days.

To determine virus shedding throat swabs were taken daily each morningprior to treatment with proanthocyanidin polymer A using a dacronnasal-pharyngeal swab. The swab was placed in 1.0 ml of tissue culture(minimum essential medium with a 10% fetal bovine serum and penicillin,streptomycin and fungizone). Titrations were performed on the throatswab solutions by preparation of ten-fold dilutions which wereinoculated into duplicate wells of BSC-40 cells grown in 24-well plates.Following incubation at 37° C. in a CO₂ incubator the titer of eachspecimen was determined by microscopic examination for viralcytopathology and the titer expressed as the log₁₀ of the TCID₅₀ per ml.At the time of each morning specimen collection each monkey was examinedfor rhinorrhea and other respiratory symptoms including coughing,sneezing and signs of respiratory distress. The animal attendant wouldalso note any coughing or sneezing during routine daily care of themonkeys. Fourteen days after virus inoculation each of the monkeys wasbled and the sera tested for antibody titers in a neutralization assayagainst approximately 100 TCID₅₀ of respiratory syncytial virus.

The results of intravenous dosing with proanthocyanidin polymer A aresummarized in Table 11. Each of the three control monkeys becameinfected with respiratory syncytial virus, shedding virus in theoropharynx for 7 to 10 days or longer. Virus titers reached five and sixlogs₁₀ in two of the three control monkeys and 3.5 log₁₀ in the third.In the monkeys receiving proanthocyanidin polymer A at the highest doseof 1 mg/kg/day, the time of virus shedding and titers of virus shed weremuch reduced. One monkey shed virus for one day, a second for threedays, and a third for four days. In one monkey, the maximum virus titerwas two logs. The mid dose of 0.5 mg/kg/day was also seen to shorten thetime of shedding in two monkeys with the third monkey excreting virus inthe oropharynx for nine days. Virus titers were generally lower althoughmaximum titers of four logs were detected in two of the three monkeysand three logs in the third. A minimal effect was seen at the lowestdose of 0.2 mg/kg/day. Virus shedding was seen for six or seven days anda maximum titers of three logs₁₀ were seen in two monkeys and five logsin the third monkey. Antibody titers to respiratory syncytial virus weredetected in all monkeys at 14 days with titers of 1:40 to 1:60.

Daily mean log₁₀ titers are presented for each treatment group (SeeTable 12). Mean titers were lower in each of the three treatment groupswhen compared with the mean titers in the control group on each of thesampling days. The mean titers were considerably reduced in the 1.0mg/kg/day treatment group, and a dose related reduction is also seen inthe other two groups.

Clinical symptoms were more prevalent in the control monkeys than in themonkeys treated with proanthocyanidin polymer A (See Table 13).Rhinorrhea is the most easily observed symptom and was seen in each ofthe three controls. No signs of toxicity were observed during dailyexaminations of the monkeys at the time of sampling or during dailyinspection during the course of the study.

                                      TABLE 11                                    __________________________________________________________________________    Effect of Intravenous Dosing With                                             Proanthocyanidin Polymer A Upon the Development of                            Respiratory Syncytial Virus Infection in African Green                        Monkeys.                                                                                                                  AB                                                                            Titer                                                                         14                                Treatment                                                                           Mnky                                                                              Log.sub.10 of Titer From Throat Swabs - Days Post-Infection                   (P.I.)                            Days                              Group #   1  2  3  4   5   6   7  8   9  10 P.I.                              __________________________________________________________________________    Control                                                                             K349                                                                              <1.0                                                                             <1.0                                                                              1.0                                                                             2.5 2.0 3.0 3.5                                                                              2.5  1.0                                                                             <1.0                                                                             1:80                              0.5% Dex.                                                                           K553                                                                              <1.0                                                                              1.0                                                                              3.0                                                                             4.0 5.0 5.0 6.0                                                                              4.5  2.0                                                                              0.5                                                                             1:160                             b.i.d., i.v.                                                                        K340                                                                              <1.0                                                                              2.5                                                                              3.0                                                                             4.0 5.0 4.0 4.0                                                                              5.0  3.0                                                                              3.0                                                                             1:160                             0.2   K152                                                                              <1.0                                                                             <1.0                                                                             <1.0                                                                             2.0 1.5 3.0 2.5                                                                              2.5  1.0                                                                              2.0                                                                             1:40                              mg/kg/day                                                                           H549                                                                              <1.0                                                                             <1.0                                                                              3.0                                                                             3.0 4.0 5.0 4.0                                                                              3.0 <1.0                                                                             <1.0                                                                             1:80                              b.i.d., i.v.                                                                        K100                                                                              <1.0                                                                             <1.0                                                                             <1.0                                                                             1.5 1.0 3.0 2.0                                                                              1.0  0.5                                                                             <1.0                                                                             1:80                              0.5   K154                                                                              <1.0                                                                             <1.0                                                                             <1.0                                                                             <1.0                                                                              2.0 1.5 3.0                                                                              4.0 <1.0                                                                             <1.0                                                                             1:40                              mg/kg/day                                                                           K332                                                                              <1.0                                                                              0.5                                                                              1.0                                                                             2.0 3.0 3.0 3.0                                                                              4.0  3.0                                                                              1.0                                                                             1:80                              b.i.d., i.v.                                                                        K155                                                                              <1.0                                                                             <1.0                                                                             <1.0                                                                             1.5 3.0 2.0 2.0                                                                              2.5 <1.0                                                                             <1.0                                                                             1:80                              1.0   K153                                                                              <1.0                                                                             <1.0                                                                             <1.0                                                                             <1.0                                                                              <1.0                                                                              <1.0                                                                              1.0                                                                              <1.0                                                                              <1.0                                                                             <1.0                                                                             1:160                             mg/kg/day                                                                           K113                                                                              <1.0                                                                             <1.0                                                                             <1.0                                                                             <1.0                                                                              1.0 1.0 2.0                                                                              1.0 <1.0                                                                             <1.0                                                                             1:160                             b.i.d., i.v.                                                                        K350                                                                              <1.0                                                                             <1.0                                                                             <1.0                                                                             <1.0                                                                              <1.0                                                                              <1.0                                                                              0.5                                                                              1.5 <1.0                                                                             <1.0                                                                             1:80                              __________________________________________________________________________

                                      TABLE 12                                    __________________________________________________________________________    Mean Titers of RSV in Throat Swabs From                                       African Green Monkeys Treated With Proanthocyanidin                           Polymer A.                                                                    Treatment                                                                             Log.sub.10 Mean Titer of RSV in Throat Swabs (Days P.I.) N = 3        Group   1  2  3   4   5   6   7   8   9   10                                  __________________________________________________________________________    Control <1.0                                                                              1.2                                                                             2.3 3.5 4.0 4.0 4.5 4.0 2.0 1.2                                 0.2 mg/kg/day                                                                         <1.0                                                                             <1.0                                                                             1.0 2.2 2.2 3.7 2.8 2.2 0.5 0.7                                 0.5 mg/kg/day                                                                         <1.0                                                                              0.2                                                                             0.3 1.2 2.7 2.2 3.0 3.2 1.0 0.3                                 1.0 mg/kg/day                                                                         <1.0                                                                             <1.0                                                                             <1.0                                                                              <1.0                                                                              0.3 0.7 1.2 0.8 <1.0                                                                              <1.0                                __________________________________________________________________________

                  TABLE 13                                                        ______________________________________                                        Symptoms of Respiratory Syncytial Virus                                       Infection in African Green Monkeys Treated With                               Proanthocyanidin Polymer A.                                                   Treatment Monkey    Days of Symptoms During 10 Days                           Group     Number    Rhinorrhea                                                                              Sneezing                                                                              Coughing                                ______________________________________                                        Control   K349      1         --      1                                       0.5% Dex  H553      2         3       --                                      b.i.d., i.v.                                                                            K340      2         6       1                                       0.2 mg/kg/day                                                                           K152      2         --      --                                      b.i.d., i.v.                                                                            K549      1         3       --                                                K100      --        --      --                                      0.5 mg/kg/day                                                                           K154      --        1       --                                      b.i.d., i.v.                                                                            K332      --        --      --                                                K155      --        --      --                                      1.0 mg/kg/day                                                                           K153      2         --      --                                      b.i.d., i.v.                                                                            K113      --        --      --                                                K350      --        --      --                                      ______________________________________                                    

In summary, proanthocyanidin polymer A was seen to have a definitebeneficial effect on respiratory syncytial virus infection in Africangreen monkeys. A dose of 1.0 mg/kg given by intravenous injection twicedaily resulted in shorter duration of virus shedding from theoropharynx, reduced titers of virus shed and reduced clinical symptoms.A dose of 0.5 mg/kg administered twice daily was also effective inreducing all three parameters of infection although to a lesser degreethan was observed at the higher dose.

9. EFFECTIVENESS OF PROANTHOCYANIDIN POLYMER A FOR TREATMENT OFINFLUENZA A

Male and female BALB/c mice were used in in vivo tests to study theeffectiveness of proanthocyanidin polymer A against Influenza A. Themice weighed 13-16 g and were obtained from Simonsen Laboratories(Gilroy, Calif.). They were quarantined 24 hr prior to use, andmaintained on Wayne Lab Blox and tap water. Once the animals wereinfected, 0.006% oxytetracycline (Pfizer, New York, N.Y.) was added tothe drinking water to control possible secondary bacterial infections.

Influenza A/NWS/33 (H1N1) was obtained from K. W. Cochran, University ofMichigan (Ann Arbor, Mich.). A virus pool was prepared by infectingconfluent monolayers of Madin Darby canine kidney (MDCK) cells,incubating them at 37° C. in 5% CO₂, and harvesting the cells at 3 to 5days when the viral cytopathic effect was 90 to 100%. The virus stockwas ampuled and stored at -80° C. until used.

The proanthocyanidin polymer A was dissolved in sterile water forinjection (WFI) each day at the appropriate concentrations and usedimmediately.1-Adamantanamine HCL (amantadine) was purchased from Sigma(St. Louis, Mo.) and was dissolved in sterile saline.1-β-D-Ribofuranosyl-1,2,4-triazole-3-carboxamide (ribavirin) waspurchased from ICN Pharmaceuticals, Inc. (Costa Mesa, Calif.) and wasdissolved in sterile saline.

An Ohmeda Blox 3740 pulse oximeter (Ohmeda, Louisville, Ohio) with earprobe attachment was used.

Each mouse lung was homogenized and varying dilutions assayed intriplicate for infectious virus in MDCK cells as described by Sidwell,R. W., Huffman J. H., Call E. W., Alghamandan H., Cook P. D., Robins, R.K. "Effect of Selenazofurin on Influenza A and B Virus Infections inMice", Antiviral Res., 6:343-53 (1986).

Two-experiments were conducted as follows: Experiment 1

Seventeen male and seventeen female mice were infected intranasally(i.n.) with an approximate 90% lethal dose of virus (0.06 ml). Thisviral inoculum was equivalent to an approximately 10⁵ cell culture 50%infectious doses in MDCK cells. Treatment with 30, 10 or 3 mg/kg/day ofproanthocyanidin polymer A or 125 mg/kg/day of amantadine was begun 2days pre-virus exposure and continued once daily for 8 days. Pulseoximeter readings of arterial oxygen saturation (SaO₂) were determinedon 5 male and 5 female mice daily through 10 days post-virus exposureand again on days 14 and 21. On infection days 2,4,6,8,10,14 and 21, 3mice (2 male, 1 female or 2 female, 1 male) were killed and their lungsremoved, graded for consolidation, and frozen for laser assay of virustiter. Consolidation was scored from 0 (normal) to 4 (100%consolidation). The same animals used for SaO₂ determinations were heldfor 21 days and deaths noted as they occurred. As toxicity controls, 3male and 3 female sham-infected mice were treated concomitantly witheach dosage level of test compound. These animals together with normaluntreated mice were weighed immediately prior to treatment and 18 hrafter the final treatment.

Experiment 2

This experiment was identical to Experiment 1, except therapy began 4 hrpost-virus exposure.

Statistical Evaluation

Increase in survivor number was evaluated using chi square analysis withYate's correction. Mean survival time increases, virus titer and SaO₂value differences were analyzed by t-test. Lung consolidation scoreswere evaluated by ranked sum analysis. The Exstatix.sup.™ program run ona Macintosh II computer was used to determine±standard errors (S.E.).

An overview of the results of these experiments is shown in Tables 14and 15. The overview of the two experiments shows mean data compiledfrom all time points in which assays were made. The toxicity controlsfor these experiments indicated the 30 mg/kg/day dose ofproanthocyanidin polymer A was toxic to the mice.

Experiment 1 (Early treatment initiation)

The summary of Experiment 1 (Table 14) indicates significant influenzadisease-inhibitory effects of the 30 and 10 mg/kg/day dosages ofproanthocyanidin polymer A based on the reduction of lung consolidation.The SaO₂ % were significantly increased in the infected female micereceiving the 10 and 3 mg/kg/day doses.

Amantadine was intended as a positive control in these experiments.Amantadine did not appear significantly effective in this experiment.However, it has been subsequently learned that this particular influenzavirus is resistant to Amantadine.

More detailed examination of these parameters can be seen in FIGS. 6-9for Experiment 1. Lung consolidation (FIG. 6) was particularly inhibitedon the early sampling days; the consolidation gradually increased in allgroups through the remainder of the experiment, but never reached thelevel of the virus controls.

Lung virus titers (FIG. 10) were not reduced early in the study, but byday 8 and 10, significant reductions were seen in mice treated with the10 and 3 mg/kg/day dosages of proanthocyanidin polymer A.

The SaO₂ data were separated for male and female mice, since 5 animalsof each sex were used for these determinations. Essentially noinhibition of SaO₂ decline was seen in male mice treated with eithercompound (FIG. 12). The female mice appeared to respond better, however(FIG. 13), with the 10 and 3 mg/kg/day doses of proanthocyanidin polymerA inhibiting by approximately 10% of the SaO₂ decline. It should benoted that in this experiment, therapy ceased on day 6, and by the nextday, the SaO₂ in the treated animals began to decrease at a rapid rate.This suggests a need to continue therapy a few additional days.

Experiment 2 (Late treatment initiation)

The summary results of this experiment are seen in Table 15. It shouldbe noted that in this delayed treatment study all the male toxicitycontrol mice survived the high dose proanthocyanidin polymer Atreatment, although they lost considerable weight. These animals wereslightly older than those used in Experiment 1, and weighed an averageof 1 g more.

As seen in Experiment 1, no significant increase in survivors was seenin the infected, treated groups, although significant mean survival timeincreases were observed in female mice treated with 10 and 3 mg/kg/dayof proanthocyanidin polymer A (Table 15). Overall mean lung scores wereagain reduced.

The alternate-day lung consolidation findings are shown in FIG. 10.Proanthocyanidin polymer A therapy again caused inhibition ofconsolidation, although to a lesser extent than seen usingearlier-initiated therapy.

Lung virus titers (FIG. 11) were only marginally reduced byproanthocyanidin polymer A therapy.

The SaO₂ data for male and female mice are summarized in FIGS. 12 and13. As was observed in Experiment 1, the female mice tended to respondbetter to proanthocyanidin polymer A therapy than the males, withsomewhat variable, but dose-responsive inhibition of SaO₂ decline. Weconclude this material has a moderate effect against the murineinfluenza infection.

In view of amantadine's inactivity, an experiment was run using a 75mg/kg/day dose of ribavirin, which is known to be highly active againstthe influenza virus infections (Sidwell, R. W., Huffman, J. H., Hare, G.P., Allen, L. B., Witkowski, J. T., Robins, R., "Broad-SpectrumAntiviral Activity of Virazole:1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxamide", Science, 177:705-6(1972)). The drug was given i.p. twice daily for 5 days beginning 4 hrpost-virus inoculation. The same disease parameters as used in thepresent experiments were employed with the ribavirin study.

The overall results of this ribavirin experiment are shown in Table 16.The drug was not lethally toxic, although it caused moderate host weightloss. All infected, ribavirin-treated mice survived the infection. Lungconsolidation and lung virus titers were significantly reduced, and themean SaO₂ decline was inhibited. The lung consolidation, virus titer,and SaO₂ data are shown in more detail in FIGS. 14-16.

Thus, in this experiment, ribavirin exerted significant influenzainhibition.

Proanthocyanidin polymer A when given to influenza A (H1N1)-infectedmice i.p. once daily for 8 days, exerted moderate inhibition of theinfection. The material was most efficacious when treatment wasinitiated 2 days pre-virus exposure, although significant inhibition wasalso seen when therapy was delayed until 4 hr post-virus exposure. Thetoxicity controls did not survive a dose of about 30 mg/kg/day.Disease-inhibiting effects were primarily limited to 10 mg/kg/day.Amantadine was ineffective against this influenza virus strain.Ribavirin was inhibitory to the virus infection. It is concluded thatproanthocyanidin polymer A has anti-influenza virus activity.

                                      TABLE 14                                    __________________________________________________________________________    Effect of i.p. Treatment with Proanthocyanidin Polymer A on Influenza A       (HIN1) Virus Infections in Mice (Early Treatment Initiation).                 __________________________________________________________________________    Animals: 13-15 g ♂ and ♀ BALB/c Mice.                                                        Treatment Schedule: qd × 8, beginning 2                                 days pre-virus inoculation.                        Virus: Influenza A/NWS/33 virus.                                                                         Treatment Route: i.p.                              Drug Diluent: Sterile H.sub.2 O                                                                          Experiment Duration: 21 Days.                      __________________________________________________________________________                   Toxicity controls                                                                            Infected, Treated                                              Surv/   Host Wt.                                                                             Surv/ MST.sup.b                                                                             Mean                                                                              Mean Lung                                                                             Mean                          Dosage Total   Change.sup.a (g)                                                                     Total (days)  Lung                                                                              Virus Titer.sup.c                                                                     SaO.sub.2                                                                     %.sup.d               Treatment                                                                             (mg/kg/day)                                                                          ♂                                                                            ♀                                                                          ♂                                                                            ♀                                                                         ♂                                                                           ♀                                                                         ♂                                                                            ♀                                                                          Score.sup.c                                                                       (log.sub.10)                                                                          ♂                                                                           ♀           __________________________________________________________________________    Proantho-                                                                             30     0/3 0/3 --  -- 0/5                                                                              0/5                                                                              5.3 4.5  2.3*                                                                             7.0     73.1                                                                             72.6               cyanidin                                                                      Polymer A                                                                             10     3/3 3/3 2.7 0.0                                                                              0/5                                                                              0/5                                                                              8.8 10.4                                                                               1.6**                                                                            6.9     80.3                                                                              83.5*                      3     3/3 3/3 3.5 1.3                                                                              0/5                                                                              0/5                                                                              8.8 8.0 2.9 6.5     80.2                                                                              83.4*             Amantadine                                                                            125    3/3 3/3 2.9 0.3                                                                              0/5                                                                              0/5                                                                              9.3 9.0 2.4 6.7     79.6                                                                             78.1               Water   --     --  --  --  -- 0/5                                                                              0/5                                                                              10.0                                                                              9.0 3.6 6.9     82.1                                                                             80.1               Normals --     10/10                                                                             10/10                                                                             3.4 0.5                                                                              -- --         0.0                               __________________________________________________________________________     .sup.a Difference between initial weight at start of treatment and weight     18 hr following final treatment of toxicity control mice.                     .sup.b Mean survival time of mice dying on or before day 21.                  .sup.c Mean lung scores and virus titers from observations made on days 2     4, 6, 8. See FIGS. 6 and 7 for comparisons of results of all these time       points.                                                                       .sup.d Mean SaO.sub.2 % of readings made daily for 11 days. See FIG. 8 fo     comparisons of daily readings.                                                *P < 0.05                                                                     **P < 0.01                                                               

                                      TABLE 15                                    __________________________________________________________________________    Effect of i.p. Treatment with Proanthocyanidin Polymer A on Influenza A       (HIN1) Virus Infections in Mice (Early Treatment Initiation).                 __________________________________________________________________________    Animals: 14-16 g ♂ and ♀ BALB/c Mice.                                                        Treatment Schedule: qd × 8, beginning 4                                 hr post-virus inoculation.                         Virus: Influenza A/NWS/33 virus.                                                                         Treatment Route: i.p.                              Drug Diluent: Sterile H.sub.2 O                                                                          Experiment Duration: 21 Days.                      __________________________________________________________________________                   Toxicity controls                                                                            Infected, Treated                                              Surv/   Host Wt.                                                                             Surv/ MST.sup.b                                                                             Mean                                                                              Mean Lung                                                                             Mean                          Dosage Total   Change.sup.a (g)                                                                     Total (days)  Lung                                                                              Virus Titer.sup.c                                                                     SaO.sub.2                                                                     %.sup.d               Compound                                                                              (mg/kg/day)                                                                          ♂                                                                            ♀                                                                          ♂                                                                            ♀                                                                         ♂                                                                           ♀                                                                         ♂                                                                            ♀                                                                          Score.sup.c                                                                       (log.sub.10)                                                                          ♂                                                                           ♀           __________________________________________________________________________    Proantho-                                                                             30     0/3 0/3 -1.2                                                                              -- 0/5                                                                              0/5                                                                              3.6 3.4  0.4**                                                                            6.3     78.1                                                                             78.4               cyanidin                                                                      Polymer A                                                                             10     3/3 3/3 3.8 0.4                                                                              0/5                                                                              0/5                                                                              8.4  11.2**                                                                            1.5*                                                                             6.4     82.8                                                                             84.1                        3     3/3 3/3 3.3 0.9                                                                              1/5                                                                              1/5                                                                              9.3   10.3**                                                                          1.9 6.7     81.7                                                                             83.0               Amantadine                                                                            125    3/3 3/3 1.2 0.6                                                                              0/5                                                                              0/5                                                                              7.2 6.8 2.0 6.8     85.4                                                                             81.0               Water   --     --  --  --  -- 2/5                                                                              0/5                                                                              8.7 7.0 2.4 6.5     82.5                                                                             83.0               Normals --     10/10                                                                             10/10                                                                             2.8 0.0                                                                              -- --         0.0         86.8                                                                             87.0               __________________________________________________________________________     .sup.a Difference between initial weight at start of treatment and weight     18 hr following final treatment of toxicity control mice.                     .sup.b Mean survival time of mice dying on or before day 21.                  .sup.c Mean lung scores and virus titers from observations made on days 2     4, 6, 8. See FIGS. 9 and 10 for comparisons of results of all these time      points.                                                                       .sup.d Mean SaO.sub.2 % of readings made daily for 11 days. See FIG. 11       for comparisons of daily readings.                                            *P < 0.05                                                                     **P < 0.01                                                               

                                      TABLE 16                                    __________________________________________________________________________    Effect of i.p. Ribavirin Treatment on Influenza A (HIN1) Virus Infections     in Mice.                                                                      __________________________________________________________________________    Animals: 14-16 g ♀  BALB/c Mice.                                                          Treatment Schedule: bid × 5 beg. 4 hr post-virus                        inoculation.                                               Virus: Influenza A/NWS/33 virus.                                                                 Treatment Route: i.p.                                      Drug Diluent: Sterile Saline                                                                     Experiment Duration: 21 Days.                              __________________________________________________________________________                   Toxicity controls                                                                       Infected, Treated                                            Dosage Surv/                                                                             Host Wt.                                                                            Surv/                                                                              MST.sup.b                                                                          Mean                                                                              Mean Virus                                                                           Mean                            Compound                                                                              (mg/kg/day)                                                                          Total                                                                             Change.sup.a (g)                                                                    Total                                                                              (days)                                                                             Score.sup.c                                                                       Titer.sup.c (log.sub.10)                                                             SaO.sub.2 %.sup.d               __________________________________________________________________________    Ribavirin                                                                             75     5/5 0.6   10/10**                                                                            >21.0**                                                                             0.3**                                                                             3.9**  81**                           Saline  --     --  --    1/18 5.8  2.3 7.3    71                              Normals --     5/5 0.6   --   --   0.0 0.0    88                              __________________________________________________________________________     .sup.a Difference between initial weight at start of treatment and weight     18 hr following final treatment of toxicity control mice.                     .sup.b Mean survival time of mice dying on or before day 21.                  .sup.c Mean lung scores and virus titers from observations made on days 2     4, 6, 8. See FIGS. 14 and 15 for comparisons of results of all these time     points.                                                                       .sup.d Mean SaO.sub.2 % of readings made daily for 10 days. See FIG. 16       for comparisons of daily readings.                                            **P < 0.01                                                               

10. EFFECTIVENESS OF PROANTHOCYANIDIN POLYMER A FOR TREATMENT OFPARAINFLUENZA VIRUS

Twelve African green monkeys without antibody to parainfluenzavirus,type 3, (PIV-3) were used in an experiment to determine antiviralactivity of proanthocyanidin polymer A. The monkeys were dividedrandomly into four groups of three monkeys each. The respective groupsreceived proanthocyanidin polymer A at doses of 10, 3.3, or 1.0mg/kg/day with the drug dissolved in 0.5% glucose. A control group ofthree monkeys received 0.5% glucose without the drug. Each of themonkeys received treatment by intravenous bolus injection as divideddoses twice daily at 8 a.m. for 7 days. The injections were made intothe saphenous vein of sedated monkeys. See Table 17.

Treatment was initiated four hours before virus inoculation. Four hourslater, a 10⁻² dilution of PIV-3 was prepared and inoculated as a 1 mlvolume intratracheally and a 1 ml volume placed on the external nares.The titer of the inoculated virus was 10³.5 TCID₅₀ per ml.

Throat swabs were taken daily and placed in 1 ml of tissue culturemedium. Fluid was expressed from the swab and the tissue culture fluidwas titrated for virus. Tenfold dilutions of the throat swab specimenswere prepared and inoculated into duplicate wells of 24 well tissueculture plates seeded with Vero cells. Titers were determined bymicroscopic examination of the cultures for viral cytopathology. Themonkeys were observed daily for clinical symptoms including rhinorrhea,sneezing and coughing. At 14 and 21 days post-infection, blood was drawnfor detection of antibody to PIV-3 using a serum neutralization assay.

Each of the 12 monkeys became infected with PIV-3 with the majorityshedding virus within 24 hours after virus inoculation. Virus sheddingcontinued in all monkeys through the eighth day post-infection with somemonkeys shedding virus through day 9 and 10. See Table 18.

The mean virus titers for each group are shown in Table 19.Proanthocyanidin polymer A at 10 mg/kg/day appear to reduce the meantiters on all of the days post-infection by one log or better. The 3.3mg/kg/day dose was seen to have a slightly lesser effect than the 10mg/kg/day dose but again all titers were less than the controls. Noappreciable effect was seen at 1.0 mg/kg/day.

Symptoms of respiratory infection were more common in the controlmonkeys than in any one of the three proanthocyanidin polymer A treatedgroups. During the 11 days of observation with 3 monkeys per group, 33total symptom days are possible. In the group receiving proanthocyanidinpolymer A at 10 mg/kg/day only 7 of those 33 days were observed to havemonkeys with symptoms. The 3.3 mg/kg/day dose resulted in only 2 daysand the 1.0 mg/kg/dose resulted in 7 days. The principal symptomobserved was rhinorrhea (See Table 20).

Antibody titers to PIV-3 at 14 and 21 days after virus inoculation arereported. All monkeys were observed to develop antibody with whatappeared to be slightly higher titers in the treated monkeys whencompared with the untreated controls. See Table 21.

Thus, proanthocyanidin polymer A was seen to have a beneficial effect onthe course of parainfluenzavirus, type 3, in African green monkeystreated with 10 and 3.3 mg/kg/day with proanthocyanidin polymer A.Proanthocyanidin polymer A was seen to reduce the titers of virus shedin the throats of infected monkeys and may have reduced the symptomsseen with this infection.

                  TABLE 17                                                        ______________________________________                                        Treatment Groups of African Green Monkeys                                     Selected to Evaluate the Antiviral Activity of                                Proanthocyanidin Polymer A* Against Parainfluenzavirus,                       Type 3                                                                        Monkey                                                                        Number  Sex    Weight (kg) Treatment with *P. Pol. A                          ______________________________________                                        L299    F      2.0         5 mg/kg, b.i.d., i.v.                              L341    M      4.6         5 mg/kg, b.i.d., i.v.                              L300    M      1.8         5 mg/kg, b.i.d., i.v.                              K144    M      5.0         1.67 mg/kg, b.i.d., i.v.                           L302    M      2.4         1.67 mg/kg, b.i.d., i.v.                           K143    M      4.6         1.67 mg/kg, b.i.d., i.v.                           L301    M      2.8         0.5 mg/kg, b.i.d., i.v.                            K347    M      4.8         0.5 mg/kg, b.i.d., i.v.                            K342    F      2.8         0.5 mg/kg, b.i.d., i.v.                            K337    F      2.8         0.5% glucose, b.i.d, iv                            K346    F      3.0         0.5% glucose, b.i.d, iv                            K345    F      3.2         0.5% glucose, b.i.d, iv                            ______________________________________                                    

                                      TABLE 18                                    __________________________________________________________________________    Effect of Proanthocyanidin Polymer A*                                         Treatment Upon Virus Shedding From African Green                              Monkeys Infected With Parainfluenzavirus, Type 3                                      Monkey                                                                             Log of Virus Titer on Days Post-Infection                        Treatment                                                                             Number                                                                             1   2   3   4   5   6   7   8   9   10                           __________________________________________________________________________     Control                                                                              K337 1.0 4.0 5.0 5.0 6.0 5.0 4.0 3.0 1.5 1.0                           0.5%   K346 2.0 3.5 5.0 4.5 5.5 6.0 5.0 4.0 2.0 1.0                           glucose iv.                                                                          K345 2.0 3.0 4.5 4.5 6.0 5.0 5.0 3.0 1.0 <1.0                         *P. Pol. A;                                                                           L299 1.0 2.0 3.0 4.0 4.0 5.0 2.5 1.5 <1.0                                                                              <1.0                          10 mg/kg/dy                                                                          K341 <1.0                                                                              2.0 3.0 4.5 3.0 3.5 3.0 2.0 1.5 <1.0                          i.v., BID                                                                            L300 1.0 2.0 3.0 4.0 3.5 4.0 3.0 1.0 <1.0                                                                              <1.0                         *P. Pol. A;                                                                           K144 <1.0                                                                              1.0 3.0 4.0 4.0 4.5 5.0 4.0 2.0 1.0                           3.3 mg/kg/dy                                                                         L302 2.0 3.0 2.0 4.5 4.0 5.0 3.5 3.0 1.0 <1.0                          i.v., BID                                                                            K143 1.0 3.0 2.5 3.5 4.5 5.0 3.5 2.0 1.0 <1.0                         *P. Pol. A;                                                                           L301 2.0 4.0 4.5 5.0 4.5 4.5 3.5 2.0 1.0 <1.0                          1.0 mg/kg/dy                                                                         K347 2.0 3.0 3.0 5.0 5.0 6.0 3.0 1.0 <1.0                                                                              <1.0                          i.v., BID                                                                            K342 2.0 3.0 4.5 5.0 5.0 6.0 5.0 3.0 1.5 1.0                          __________________________________________________________________________

                  TABLE 19                                                        ______________________________________                                        Mean Daily Titers of Parainfluenzavirus,                                      Type 3 in African Green Monkeys Treated With                                  Proanthocyanidin Polymer A                                                            Mean Log of Virus Titer on Days Post-Infection                        Treatment 1     2     3    4   5   6   7   8   9   10                         ______________________________________                                         Glucose  1.7   3.5   4.8  4.7 5.8 5.5 4.7 3.3 1.5 0.7                         Control                                                                      *P. Polymer A;                                                                          0.7   2.0   3.0  4.2 3.5 4.2 2.8 1.5 0.5 <1.0                        10 mg/kg/day                                                                 *P. Polymer A;                                                                          1.0   2.3    2.5.                                                                              4.0 4.2 4.8 4.0 3.0 1.3 0.3                         3.3 mg/kg/day                                                                *P. Polymer A;                                                                          2.0   3.3   4.0  5.0 4.8 5.5 4.2 2.0 0.8 0.3                         1.0 mg/kg/day                                                                ______________________________________                                    

                  TABLE 20                                                        ______________________________________                                        Total Days in Which Monkeys Showed Symptoms                                   to Parainfluenzavirus, Type 3 Infection                                       Treatment                                                                     Days/Total          Symptom                                                   ______________________________________                                        Control, Glucose    13/33                                                     P. Pol. A, 10 mg/kg/day                                                                            7/33                                                     P. Pol. A, 3.3 mg/kg/day                                                                           2/33                                                     P. Pol. A, 1.0 mg/kg/day                                                                           7/33                                                     ______________________________________                                    

                  TABLE 21                                                        ______________________________________                                        Antibody Titers to Parainfluenzavirus, Type                                   3, in African Green Monkeys Infected With the Virus                           and Treated With Proanthocyanidin Polymer A*                                  P.I.                     Antibody                                             Treatment   Monkey        Titer at Days                                       Days        Number       14 Days  21                                          ______________________________________                                         Control    K337         1:40     1:160                                        0.5%       K346         1:20     1:80                                         glucose, i.v.                                                                            K345         1:80     1:160                                       *P. Polymer A,                                                                            L299         1:160    1:320                                        10 mg/kg/day,                                                                            K341         1:160    1:320                                        i.v.       L300         1:160    1:320                                       *P. Polymer A,                                                                            K144         1:80     1:80                                         3.3 mg/kg/day,                                                                           L302         1:160    1:320                                        i.v.       K143         1:160    1:160                                       *P. Polymer A,                                                                            L301         1:160    1:320                                        1.0 mg/kg/day                                                                            K347         1:80     1:160                                        i.v.       K342         1:40     1:180                                       ______________________________________                                    

10.1 EFFECTIVENESS OF PROANTHOCYANIDIN POLYMER A FOR TREATMENT OF HERPESSIMPLEX VIRUS TYPE 2 VAGINITIS

In vivo experiments were performed to study the effectiveness ofproanthocyanidin polymer A in comparison with ganciclovir against herpessimplex virus type 2. Ganciclovir is known to be effective againstherpes simplex virus type 2.

In one series of experiments, both compositions were formulated forintraperitoneal injection by dissolving in physiological saline.

Swiss Webster female mice (Simonsen Labs, Gilroy, Calif.) weighingapproximately 20 grams each at the start of the experiment were infectedintravaginally with herpes simplex virus type 2 (HSV-2), E194 strain.This was accomplished in a 3-step process. First, the vagina of eachmouse was swabbed for 5 seconds with a cotton tip applicator dipped in0.1N NaOH. This treatment irritates the vaginal area so that theinfection takes better. Approximately 1 hour later each vagina was dryswabbed for 5 seconds. Then an applicator dipped in virus medium wasused to swab each mouse for 20 seconds. The swabs were gently and slowlytwisted back and forth during the time they were in place.

Six hours after virus infection, intraperitoneal (i.p.) injections wereadministered using proanthocyanidin polymer A, ganciclovir, or placebo.Treatments were also given i.p. twice daily on days 1 through 7 aftervirus challenge. The daily dose of each compound was 30 mg/kg/day, with15 mg/kg given at each injection. Since on day 0 of the infection onlyone dose was given, the daily dose for that day was 15 mg/kg/day.

Groups of 5 mice were sham-infected using the process described abovefor virus infection, except that no virus was present for the finalstep. These mice were treated the same way and at the same times asabove. Mice were checked daily for survival, and weights were recordedbefore the first (day 0) and after the last (day 8) treatment.

Lesion scores in infected mice were determined daily on days 3-14 of theinfection. A score of 1+ indicates redness immediately around thevagina. 2+ indicates spread of the lesion toward the anus. 3+ indicatesa lesion (usually with swelling) from the vagina to the anus. There arevariations to this since some mice may have a vaginal lesion plus alesion on the tail. Because many of the mice go on to die, the lesionscore near the time of death is carried through to the end of the 14days. If this were not done, lesion scores in the placebo group wouldappear to go down as the most affected mice die off. Some animalsdeveloped hind limb paralysis (and later died). This condition did notadd to the lesion score.

Deaths were recorded daily for 21 days. The mean day of deathcalculation took into account only mice that died. Vaginal virus titerswere made by titration of virus obtained from vaginal swabs 3 days aftervirus inoculation. These titrations were conducted in Vero cells in96-well plates. Calculation of virus titer was made by the 50% endpointdilution method of Reed, L. J. and Meunch, M., Am. J. Hyg., 27:493-498(1938).

Statistical interpretations of survival (Fisher exact test), mean day todeath (Student's t test) and virus titer (Student's t test) were made bytwo-tailed analyses.

Table 22 below shows the average lesion scores with standard deviationsand statistical analyses for the experiment.

                  TABLE 22                                                        ______________________________________                                        Effect of Intraperitoneal Proanthocyanidin                                    Polymer A Treatment on Herpes Simplex Virus Type 2                            (HSV-2) Vaginitis in Mice.                                                            Average Lesion Score                                                                        Ganciclovir                                                       Proanth. Pol. A                                                                           (30 mg/kg/day)                                          Day.sup.a (30 mg/kg/day                                                                             Placebo                                                 ______________________________________                                         3        .sup. 0.2 ± 0.2.sup.b                                                                  0.1 ± 0.2  0.3 ± 0.3                               4        0.2 ± 0.3                                                                              0.2 ± 0.2**                                                                              1.1 ± 1.0                               5         0.7 ± 0.9**                                                                           0.2 ± 0.2**                                                                              1.9 ± 1.2                               6         1.3 ± 1.4**                                                                           0.2 ± 0.2***                                                                             3.1 ± 1.4                               7         1.5 ± 1.3**                                                                           0.2 ± 0.2***                                                                             3.1 ± 1.6                               8         1.8 ± 1.5*                                                                            0.3 ± 0.4***                                                                             2.0 ± 1.6                               9        2.1 ± 1.4                                                                              0.4 ± 0.3***                                                                             3.2 ± 1.4                              10        2.3 ± 1.4                                                                              0.5 ± 0.5***                                                                             3.2 ± 1.3                              11        2.3 ± 1.5                                                        .4***     3.2 ± 1.30.5 ±                                                12        2.2 ± 1.4                                                                              0.9 ± 0.2***                                                                             3.2 ± 1.3                              13         1.8 ± 1.5*                                                                            0.6 ± 0.6***                                                                             3.2 ± 1.4                              14         1.8 ± 1.5*                                                                            0.6 ± 0.6***                                                                             3.1 ± 1.4                              Grand Avg.                                                                               1.5 ± 0.8**                                                                           0.4 ± 0.2***                                                                             2.5 ± 1.0                              (Days 3-14)                                                                   ______________________________________                                         .sup.a After virus challenge.                                                 .sup.b Standard deviation.                                                    *p < 0.05,                                                                    **p < 0.01                                                                    ***p < 0.001 (twotailed Student's .sub.- t test).                        

Proanthocyanidin polymer A showed a statistically significant decreasein average lesion score compared to the placebo control. Ganciclovir wasalso effective. FIG. 17 gives a visual impression of the degree oflesion inhibition exhibited by 30 mg proanthocyanidin polymer A per kgrelative to the placebo control and ganciclovir. Table 23 shows survivalof the treated mice.

                  TABLE 23                                                        ______________________________________                                        Effect of Intraperitoneal Proanthocyanidin                                    Polymer A Treatment on Survival and Vaginal Virus                             Titer in Mice Infected Intravaginally with Herpes                             Simplex Virus Type 2 (HSV-2).                                                 ______________________________________                                                        Survivors/                                                                              Mean Day                                                                              Virus Titer.sup.a                           Compound Dose   Total (%) to Death                                                                              (Day 3)                                     ______________________________________                                        Virus-Infected                                                                Pro. Pol. A                                                                            30      4/10(40) 9.5 ± 3.2                                                                          2.8 ± 1.1                                Ganciclovir                                                                            30     10/10(100)**                                                                            >21      1.7 ± 1.0*                              Placebo  --      3/20(15) 8.2 ± 2.9                                                                          3.8 ± 1.2                                Uninfected Toxicity Controls                                                                                    Host Wt. Change.sup.c                       Pro. Pol. A                                                                            30      5/5(100) >21     +2.8                                        Ganciclovir                                                                            30      5/5(100) >21     +1.0                                        Placebo  --      5/5(100) >21     +2.5                                        ______________________________________                                         .sup.a Log.sub.10 50% cell culture infectious dose per ml.                    .sup.b Determined on day 8. Represents the difference in grams between da     0 and day 8 weights.                                                          *P < 0.002, twotailed Fisher exact test (survival) or twotailed Student's     t test (mean day to death and virus titer parameters.)                   

Proanthocyanidin polymer A showed an increased survival (40% survivalcompared to 15% in the placebo group). The ganciclovir group had 100%survival. Table 23 also shows that proanthocyanidin polymer A treatedmice had less virus than the placebo control, but the results were notstatistically significant. Ganciclovir caused a significant reduction invirus titer.

Results in toxicity control mice showed proanthocyanidin polymer A waswell tolerated and caused no adverse effects at the doses used.Ganciclovir treated mice gained less weight than placebo controls.

Thus, proanthocyanidin polymer A administered intraperitoneally at adose of 30 mg/kg/day had moderate antiviral activity in this model.There was no apparent toxicity of the proanthocyanidin polymer Aformulation to mice.

Another series of preliminary experiments, in which the proanthocyanidinpolymer A composition is administered orally, showed some anti-HSV-2activity but only at the highest dose administered, i.e., 270 mg/kg/day.

In another series of experiments, the proanthocyanidin polymer Acomposition was formulated for topical application against HSV-2. Theefficacy of proanthocyanidin polymer A is compared with ganciclovir andacyclovir. Both ganciclovir powder and acyclovir cream were obtainedcommercially. Squibb cream base #8, which served as a vehicle and as thetopical placebo control, was also commercially obtained.

The methods of infecting the mice, setting toxicity control and theparameters measured to evaluate the infection were the same as describedabove for the intraperitoneal tests. Topical treatments on the day ofinfection were given at +6 hours. They were then given twice daily for 7more days.

Treatment for 7.5 days with proanthocyanidin polymer A at two dosesresulted in a statistically significant reduction in lesion scores ofthe period of evaluation for the 10% dose. This is presented graphicallyin FIG. 18 and in Table 24. The 5% dose also reduced lesion developmentrelative to the placebo control, but the results were not statisticallysignificant. Acyclovir cream nearly completely prevented lesionformation in infected animals. More mice treated with proanthocyanidinpolymer A at 5% and 10% doses survived the infection that placebocontrols (Table 25) and vaginal virus titers were reduced at the 10%dose. In addition, the numbers of mice without lesions on day 14 weregreater than in the placebo group. None of the uninfected toxicitycontrol mice lost weight or died as a result of treatment with any ofthe cream formulations. (Table 25).

It can be concluded that topically applied proanthocyanidin polymer A asa 10% cream was active. Only three mice developed vaginal lesions, butthe lesions all progressed to maximum severity. The proanthocyanidinpolymer A cream formulations were somewhat dry to the touch, since thecomposition hydrated and absorbed some of the water from the Squibbcream base. This may have affected the performance of theproanthocyanidin polymer A cream. Partial hydration of the material inwater prior to mixing it with the cream base may provide a more aqueousand bioavailable product for topical application.

                  TABLE 24                                                        ______________________________________                                        Effect of topical Proanthocyanidin Polymer                                    A Treatment on Herpes Simplex Virus Type 2 (HSV-2)                            Vaginitis in Mice.                                                                   Average Lesion Score                                                            P. Pol.   P. Pol.   Acyclovir                                        Day.sup.a                                                                              A 5%      A 10%     5%       Placebo                                 ______________________________________                                         3        0.3 ± 0.3.sup.b                                                                     0.2 ± 0.3                                                                            0.2 ± 0.2                                                                           0.3 ± 0.3                             4       0.3 ± 0.5                                                                            0.6 ± 1.0                                                                            0.1 ± 0.2                                                                           0.5 ± 0.9                             5       0.7 ± 1.1                                                                            0.6 ± 1.1                                                                            0.0 ± 0.0***                                                                        0.8 ± 0.9                             6       1.0 ± 1.6                                                                            1.3 ± 1.7                                                                            0.0 ± 0.0***                                                                        1.7 ± 1.5                             7       1.7 ± 1.8                                                                            1.1 ± 1.8                                                                            0.0 ± 0.0***                                                                        2.3 ± 1.7                             8       1.9 ± 2.0                                                                            1.1 ± 1.8                                                                            0.1 ± 0.2***                                                                        2.5 ± 1.8                             9       1.9 ± 1.8                                                                            1.1 ± 1.8                                                                            0.0 ± 0.0***                                                                        2.6 ± 1.8                            10       1.6 ± 1.1                                                                            1.1 ± 1.8                                                                            0.0 ± 2.6 ± 1.8                            11       1.6 ± 1.1                                                                            1.1 ± 1.8                                                                            0.0 ± 0.0***                                                                        2.6 ± 1.8                            12       1.6 ± 1.1                                                                            1.1 ± 1.8                                                                            0.0 ± 0.0***                                                                        2.6 ± 1.8                            13       1.6 ± 1.1                                                                            1.1 ± 1.8                                                                            0.0 ± 0.0***                                                                        2.6 ± 1.8                            14       1.6 ± 1.1                                                                            1.1 ± 1.8                                                                            0.0 ± 0.0***                                                                        2.6 ± 1.8                            Grnd Avg 1.4 ± 0.6                                                                            1.0 ± 0.3**                                                                          0.1 ⊥ 0.1***                                                                      2.0 ± 0.9                            (Days 3-14)                                                                   ______________________________________                                         .sup.a After virus challenge.                                                 .sup.b Standard deviation.                                                    *p < 0.05,                                                                    **p < 0.01,                                                                   ***p < 0.001 (twotailed Student's t test).                               

                                      TABLE 25                                    __________________________________________________________________________    Effect of Topical Proanthocyanidin Polymer                                    A Treatment on Survival, Vaginal Virus Titers, and                            Numbers Without Lesions on Mice Infected Intra-                               vaginally with Herpes Simplex Virus Type 2 (HSV-2).                           __________________________________________________________________________              Survivors/                                                                           Mean Day                                                                            Virus Titer.sup.b                                                                      No. of Mice.sup.c                             Compound                                                                            Dose.sup.a                                                                        Total (%)                                                                            to Death                                                                            (Day 2)  W/O Lesion                                    __________________________________________________________________________    Virus-Infected                                                                Placebo                                                                             --   6/20 (30)                                                                           .sup. 9.6 ± 2.6.sup.d                                                            2.8 ± 2.2                                                                           6/20                                          Acyclovir                                                                            5  10/10 (100)*                                                                         >21    0.6 ± 0.2*                                                                         10/10                                         P. Poly A                                                                            5   5/10 (50)                                                                           9.4 ± 1.1                                                                        2.6 ± 2.5                                                                           5/10                                          P. Poly A                                                                           10   7/10 (70)                                                                           7.7 ± 1.5                                                                        1.3 ± 1.2                                                                           7/10                                          Uninfected Toxicity Controls                                                                         Host Wt. Change.sup.e                                  Placebo                                                                             --   5/5 (100)                                                                           >21   +1.3                                                   Acyclovir                                                                            5   5/5 (100)                                                                           >21   +2.0                                                   P. Poly A                                                                           10   5/5 (100)                                                                           >21   +1.6                                                   __________________________________________________________________________     .sup.a Percent of compound in a polyethylene glycol cream base.               .sup.b Log .sub.10 50% cell culture infectious dose per ml.                   .sup.c On day 14 of the infection.                                            .sup.d Standard deviation.                                                    .sup.e Determined on day 8. Represents the difference in grams between da     0 and day 8 weigh                                                             *P < 0.05, twotailed Fisher exact test (survival and numbers without          lesions) or twotailed Student's t test (mean day to death and virus titer     parameters.)                                                             

11. EFFECTIVENESS OF PROANTHOCYANIDIN POLYMER B FOR TREATMENT OF RSV

Results from in vitro tests for RSV antiviral activity, performed underthe same procedures as the tests with proanthocyanidin polymer A,indicate that proanthocyanidin polymer B is effective and has an ED₅₀ of6 μg/ml.

The invention described and claimed herein is not to be limited in scopeby the specific embodiments herein disclosed, since these embodimentsare intended as illustrations of several aspects of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims.

What is claimed is:
 1. A proanthocyanidin polymer composition,characterized by:(a) the capability of dissolving in water and/oraqueous solution; (b) the capability of exerting a pronounced antiviraleffect as demonstrated in in vitro assays for antiviral activity againstrespiratory syncytial virus, types A and B; parainfluenza virus, types 1and 3; influenza virus, types A and B; and as demonstrated in in vivotests for antiviral activity against respiratory syncytial virus;influenza virus, type A; parainfluenza virus, type 3; and herpes simplexvirus, type 2; (c) comprising a proanthocyanidin polymer having astructure comprising flavonoid units selected from the group consistingof catechins, epicatechins, gallocatechins, galloepicatechins andcombinations thereof; and (d) ¹³ C NMR spectra having peak positions atδ 154.2, 145.1, 143.7, 132.8, 131.2, 130.3, 120.9-118.6 (series of broadpeaks), 116.1, 115.4, 114.3, 108.0, 106.3, 96.6, 95.3, 81.8, 77.6, 75.3,72.6, 1.5, 65.6, 37.1, 35.3, and 27.7.
 2. The proanthocyanidin polymercomposition of claim 1 having an average of about 2 to about 11flavonoid units.
 3. The proanthocyanidin polymer composition of claim 1having an average of about 7 flavonoid.
 4. The proanthocyanidin polymercomposition of claim 1 wherein the ¹³ C NMR spectra is substantially asillustrated in FIG.
 3. 5. The proanthocyanidin polymer composition ofclaim 1 further characterized by:(a) infrared spectral analysis ofintense peaks ranging from 3350-2500 and other peaks at 1612, 1449,1348, 1202, 1144, 11076, 1068 and 1207 cm-1; (b) an ultraviolet spectrahaving broad peaks at wavelength 202, 235 (shoulder) and 275, and 305(shoulder) nm; and (c) visible spectral absorption at wavelength ofabout 460 nm.
 6. The proanthocyanidin polymer composition of claim 5wherein the infrared spectra is substantially as illustrated in FIG. 1.7. The proanthocyanidin polymer composition of claim 1 obtained from aCroton species.
 8. The proanthocyanidin polymer composition of claim 7obtained from Croton lechleri.
 9. An ester, ether or oxonium derivativeof a proanthocyanidin polymer, in which the polymer is characterizedby:(a) the capability of dissolving in water and/or aqueous solution;(b) the capability of exerting a pronounced antiviral effect asdemonstrated in in vitro assays for antiviral activity againstrespiratory syncytial virus, types A and B; parainfluenza virus, types 1and 3; influenza virus, types A and B; and as demonstrated in in vivotests for antiviral activity against respiratory syncytial virus;influenza virus, type A; parainfluenza virus, type 3; and herpes simplexvirus, type 2; (c) having a structure comprising flavonoid unitsselected from the group consisting of catechins, epicatechins,gallocatechins, galloepicatechins and combinations thereof; and (d) ¹³ CNMR spectra having peak positions at δ 154.2, 145.1, 143.7, 132.8,131.2, 130.3, 120.9-118.6 (series of broad peaks), 116.1, 115.4, 114.3,108.0, 106.3, 96.6, 95.3, 81.8, 77.6, 75.3, 72.6, 71.5, 65.6, 37.1,35.3, and 27.7.
 10. A proanthocyanidin polymer composition characterizedby:(a) the capability of dissolving in water and/or an aqueous solution;(b) the capability of exerting an antiviral effect as demonstrated in invitro assays for antiviral activity against respiratory syncytial virus,types A and B; parainfluenza virus, types 1 and 3; influenza virus,types A and B; and as demonstrated in in vivo tests for antiviralactivity against respiratory syncytial virus; influenza virus, type A;parainfluenza virus, type 3; and herpes simplex virus, type 2; (c)comprising a proanthocyanidin polymer having a structure comprisingflavonoid units selected from the group consisting of catechins andepicatechins; and (d) a ¹³ C NMR spectra substantially as illustrated inFIG.
 4. 11. The proanthocyanidin polymer composition of claim 10 furthercharacterized by an infrared spectra substantially as illustrated inFIG. 5; and ultraviolet spectra having peaks at wavelength 202-204 and275-280 nm.
 12. The proanthocyanidin polymer composition of claim 10which is obtained from Calophyllum inophylum.
 13. An ester, ether oroxonium derivative of a proanthocyanidin polymer, in which the polymeris characterized by:(a) the capability of dissolving in water and/oraqueous solution; (b) the capability of exerting an antiviral effect asdemonstrated in in vitro assays for antiviral activity againstrespiratory syncytial virus, types A and B; parainfluenza virus, types 1and 3; influenza virus, types A and B; and as demonstrated in in vivotests for antiviral activity against respiratory syncytial virus;influenza virus, type A; parainfluenza virus, type 3; and herpes simplexvirus, type 2; (c) comprising a proanthocyanidin polymer having astructure comprising flavonoid units selected from the group consistingof catechins and epicatechins; and (d) a ¹³ C NMR spectra substantiallyas illustrated in FIG.
 4. 14. A proanthocyanidin polymer compositionobtained from a Croton tree by a method which comprises:(a) extractingthe whole plane, the bark, the stems, the roots or the latex of theCroton tree with a lower alcohol of about 1-3 carbons, acetone, water orother water miscible solvent or combinations thereof to obtain anaqueous soluble fraction; (b) subjecting the aqueous soluble fraction togel filtration, ultrafiltration or chromatography using aqueous and/ororganic solvents; or combination thereof and (c) collecting the fractiondetectable by ultra violet spectroscopy having a λ maximum at about200-350 nm, wherein the collected fraction is characterized by having ¹³C NMR spectra having peak positions at δ154.2, 145.1, 143.7, 132.8,131.2, 130.3, 120.9-118.6 (series of broad peaks), 116.1, 115.4, 114.3,108.0, 106.3, 96.6, 95.3, 81.8, 77.6, 75.3, 72.6, 71.5, 65.6, 37.1,35.3, and 27.7.
 15. The composition according to claim 14, in which theCroton tree is Croton lechleri.
 16. The composition according to claim14 wherein the ¹³ C NMR spectrum is substantially as illustrated in FIG.3.
 17. The composition according to claim 14 further characterizedby:(a) infrared spectrum having intense peaks ranging from 3350-2500 andother peaks at 1612, 1449, 1348, 1202, 1144, 1107, 1068 and 1207 cm-1;(b) an ultraviolet spectra having broad peaks at wavelength 202, 235(shoulder) and 275, and 305 (shoulder); and (c) visible spectralabsorption at wavelength 460 nm.
 18. A proanthocyanidin polymercomposition obtained from Calophyllum inophylum by a method whichcomprises:(a) extracting the whole plane, the bark, the stems, the rootsor the latex of the Calophyllum inophylum with a lower alcohol of about1-3 carbons, acetone, water or other water miscible solvent orcombinations thereof to obtain an aqueous soluble fraction; (b)subjecting the aqueous soluble fraction to gel filtration,ultrafiltration or chromatography using aqueous and/or organic solvents;or combination thereof and (c) collecting the fraction detectable byultra violet spectroscopy having a λ maximum at about 200-350 nm,wherein the collected fraction is characterized by having ¹³ C NMRspectra having peak positions at δ154.2, 145.1, 143.7, 132.8, 131.2,130.3, 120.9-118.6 (series of broad peaks), 116.1, 115.4, 114.3, 108.0,106.3, 96.6, 95.3, 81.8, 77.6, 75.3, 72.6, 71.5, 65.6, 37.1, 35.3, and27.7.
 19. The composition according to claim 18 wherein the ¹³ C NMRspectrum is substantially as illustrated in FIG.
 4. 20. The compositionaccording to claim 18 further characterized by an infrared spectrasubstantially as illustrated in FIG. 5; and ultraviolet spectra havingpeaks at wavelength 202-204 and 275-280 nm.